Methods for inducing differentiation of a cell using phenyacetic acid and derivatives

ABSTRACT

Compositions and methods of treating anemia, cancer, AIDS, or severe beta -chain hemoglobinopathies by administering a therapeutically effective amount of phenylacetate or pharmaceutically acceptable derivatives thereof or derivatives thereof alone or in combination or in conjunction with other therapeutic agents. Pharmacologically-acceptable salts alone or in combinations and methods of preventing AIDS and malignant conditions, and inducing cell differentiation are also aspects of this invention.

This application is a division of application Ser. No. 08/135,661, filedon Oct. 12, 1993 which is, in turn, is a Continuation-In-Part ofApplicant's copending U.S. Ser. No. 07/779,744, filed Oct. 21, 1991, thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods of using phenylacetic acid and itspharmaceutically acceptable derivatives to treat and prevent pathologiesand to modulate cellular activities. In particular, this inventionrelates to A) phenylacetate and its derivatives in cancer prevention andmaintenance therapy, B) phenylacetate and its derivatives in thetreatment and prevention of AIDS, C) induction of fetal hemoglobinsynthesis in β-chain hemoglobinopathy by phenylacetate and itsderivatives, D) use of phenylacetic acid and its derivatives in woundhealing, E) use of phenylacetic acid and its derivatives in treatment ofdiseases associated with interleukin-6, F) use of phenylacetic acid andits derivatives in the treatment of AIDS-associated CNS dysfunction, G)use of phenylacetic acid and its derivatives to enhanceimmunosurveillance, H) methods of monitoring the dosage level ofphenylacetic acid and its derivatives in a patient and/or the patientresponse to these drugs, I) the activation of the PPAR by phenylaceticacid and its derivatives, J) use of phenylacetic acid and itsderivatives in treatment of cancers having a multiple-drug resistantphenotype, and K) phenylacetic acid and its derivatives, correlationbetween potency and lipophilicity.

BACKGROUND OF THE INVENTION

Phenylacetic acid (PAA) is a protein decomposition product foundthroughout the phylogenetic spectrum, ranging from bacteria to man.Highly conserved in evolution, PAA may play a fundamental role in growthcontrol and differentiation. In plants, PAA serves as a growth hormone(auxin) promoting cell proliferation and enlargement at low doses (10⁻⁵-10⁻⁷ M), while inhibiting growth at higher concentrations. The effecton animal and human cells is less well characterized. In humans, PAA isknown to conjugate glutamine with subsequent renal excretion ofphenylacetylglutamine (PAG). The latter, leading to waste nitrogenexcretion, has been the basis for using PAA or preferably its saltsodium phenylacetate (NaPA, also referenced herein as that activeanionic meoity, phenylacetate or "PA") in the treatment ofhyperammonemia associated with inborn errors of ureagenesis. Clinicalexperience indicates that acute or long-term treatment with high NaPAdoses is well tolerated, essentially free of adverse effects, andeffective in removing excess glutamine. [Brusilow, S. W., Horwich, A. L.Urea cycle enzymes. Metabolic Basis of Inherited Diseases, Vol.6:629-633 (1989)]. These characteristics should be of value intreatments of cancer and prevention of cancer, treatments which inhibitvirus replication and treatments of severe beta-chainhemoglobinopathies.

Glutamine is the major nitrogen source for nucleic acid and proteinsynthesis, and a substrate for energy in rapidly dividing normal andtumor cells. Compared with normal tissues, most tumors, due to decreasedsynthesis of glutamine along with accelerated utilization andcatabolism, operate at limiting levels of glutamine availability, andconsequently are sensitive to further glutamine depletion. Consideringthe imbalance in glutamine metabolism in tumor cells and the ability ofPAA to remove glutamine, PAA has been proposed as a potential antitumoragent; however, no data has previously been provided to substantiatethis proposal. [Neish, W. J. P. "Phenylacetic Acid as a PotentialTherapeutic Agent for the Treatment of Human Cancer", Experentia, Vol.27, pp. 860-861 (1971)].

Despite these efforts to fight cancer, many malignant diseases that areof interest in this application continue to present major challenges toclinical oncology. Prostate cancer, for example, is the second mostcommon cause of cancer deaths in men. Current treatment protocols relyprimarily on hormonal manipulations. However, in spite of initial highresponse rates, patients often develop hormone-refractory tumors,leading to rapid disease progression with poor prognosis. Overall, theresults of cytotoxic chemotherapy have been disappointing, indicating along felt need for new approaches to treatment of advanced prostaticcancer. Other diseases resulting from abnormal cell replication, forexample metastatic melanomas, brain tumors of glial origin (e.g.,astrocytomas), and lung adenocarcinoma, are also highly aggressivemalignancies with poor prognosis. The incidence of melanoma and lungadenocarcinoma has been increasing significantly in recent years.Surgical treatments of brain tumors often fail to remove all tumortissues, resulting in recurrences. Systemic chemotherapy is hindered byblood barriers. Therefore, there is an urgent need for new approaches tothe treatment of human malignancies including advanced prostatic cancer,melanoma, brain tumors.

The development of the methods and pharmaceuticals of the presentinvention was guided by the hypothesis that metabolic traits thatdistinguish tumors from normal cells could potentially serve as targetsfor therapeutic intervention. For instance, tumor cells show uniquerequirements for specific amino acids such as glutamine. Thus, glutaminemay be a desired choice because of its major contribution to energymetabolism and to synthesis of purines, pyrimidines, and proteins. Alongthis line, promising antineoplastic activities have been demonstratedwith glutamine-depleting enzymes such as glutaminase, and variousglutamine antimetabolites. Unfortunately, the clinical usefulness ofthese drugs has been limited by unacceptable toxicities. Consequently,the present invention focuses on PAA, a plasma component known toconjugate glutamine in vivo, and the pharmaceutically acceptablederivatives of PAA.

In addition to its ability to bind gluatamine to form glutaminephenylacetate, PAA can induce tumor cells to undergo differentiation.(See examples 1-5, 7-9, 11-13, and 16 herein). Differentiation therapyis a known, desirable approach for cancer intervention. The underlyinghypothesis is that neoplastic transformation results from defects incellular differentiation. Inducing tumor cells to differentiate wouldprevent tumor progression and bring about reversal of malignancy.Several differentiation agents are known, but their clinicalapplications have been hindered by unacceptable toxicities and/ordeleterious side effects.

Accordingly, the present invention provides methods and compositions fortreating various pathologies with PAA and its pharmaceuticallyacceptable salts, derivatives, and analogs.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of treating various pathologies in asubject. The invention also provides for the modulation of variouscellular activities in a subject. The pathologies and cellularactivities are treated and modulated utilizing a compound having theformula: ##STR1## ; wherein R₀ =aryl, phenoxy, substituted aryl orsubstituted phenoxy;

R₁ and R₂ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen;

R₃ and R₄ =H, lower alkoxy, lower straight and branched chain alkyl orhalogen; and

n=an integer from 0 to 2.

Specifically, the invention provides a method of treating or preventingvarious neoplastic conditions. Relatedly, a method of inducingdifferentiation of a cell is provided. The invention also provides amethod of inducing the production of fetal hemoglobin and treatingpathologies associated with abnormal hemoglobin activity or production.

The invention also provides a method of treating or preventing a viralinfection in a subject. Relatedly, the invention provides a method oftreating an AIDS-associated dysfunction of the central nervous system ina subject.

Also provided is a method of modulating the production of IL-6 or TGFαand TGF-β2 both in vitro and in vivo. Typically, IL-6 and TGF-β2 areinhibited while TGFα is induced.

The invention also provides a method of enhancing immunosurveillance andpromoting wound healing in a subject.

Also provided is a method of monitoring the bioavailability of acompound for treatment of a pathology not associated with hemoglobin.The method comprises administering to a subject the compound andmeasuring the level of fetal hemoglobin TGF-β2, IL-6 or TGFα.

Finally, a method of treating a neoplastic condition in cells resistantto radiation and chemotherapy is provided. Specifically, multiple drugresistant cells are particularly sensitive to the compounds of thisinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of HL-60 leukemia and premalignant 10T1/2cell proliferation by NaPA.

FIG. 2 shows the induction of HL-60 cell differentiation. The number ofNBT positive cells was determined after 4 [solid bars] or 7 days[hatched bars] of treatment. NaPA (h), 1.6 mg/ml; NaPA (1), 0.8 mg./ml.4-hydroxyphenylacetate and PAG were used at 1.6 mg./ml. Potentiation byRA 10 nM was comparable to that by IFN gamma 300 IU/ml, and the effectof acivicin 3 μg/ml similar to DON 30 μg/ml. Glutamine Starvation (Gln,<0.06 mM) was as described. Cell viability was over 95% in all cases,except for DON and acivicin (75% and 63%, respectively).

FIG. 3A shows adipocyte conversion in 10T1/2 cultures.

FIG. 4 shows NaPA's ability to invoke growth arrest of humanglioblastoma cells. Dose-dependent inhibition of human glioblastoma cellproliferation by sodium phenylacetate. Growth rates were determined,after 4-5 days of continuous treatment, by an enzymatic assay using3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertrazolium bromide andconfirmed by cell enumeration with a hemocytometer. Reduction in cellnumber paralleled changes in de novo DNA synthesis (not shown).

FIG. 5 shows selective cytostasis induced by phenylacetate (5 mM)combined with glutamine starvation (0.2 mM glutamine, i.e., 2-3 foldbelow the normal plasma levels). The results indicate increasedvulnerability of glioblastoma A172 when compared to actively replicatingnormal human umbilical vein endothelial cells (HUVC). Cell viability wasover 95% in all cases.

FIG. 6 shows that phenylacetate inhibits the mevalonate pathway ofcholesterol synthesis in glioblastoma cells. FIG. 6 shows key steps ofthe MVA pathway discussed in text.

FIG. 7 shows the selective inhibition of cholesterol synthesis frommevalonate in phenylacetate-treated glioblastoma U87 cells, andenzymatic inhibition of mevalonate decarboxylation in cell homogenates.For analysis of steroid synthesis, logarithmically growing cells werelabeled with tritiated MVA in the presence or absence of 5 mMphenylacetate, and their steroids were separated by silica thin layerchromatography. MVA decarboxylation was measured in cell homogenates.The effect of phenylacetate on cholesterol synthesis and MVAdecarboxylation was selective as, under the experimental conditionsused, total protein and DNA synthesis levels were unaffected.

FIG. 8 shows the effects of phenylacetate on rate of proliferation afterin vitro exposure of 9L tumor cells to various concentrations ofphenylacetate for 5 days. Significant decline in DNA-synthesis wasobserved. Data are expressed as means±S.D. counts per minute (cpm).

FIG. 9 shows the treatment with phenylacetate from the day ofintracerebral tumor inoculation extended survival compared withtreatment with saline (p<0.01; Mantel-Haenzel test).

FIG. 10 shows the treatment of established tumors with phenylacetateextended survival compared to treatment with saline (p<0.03;Mantel-Haenzel test).

FIG. 11 shows the effect of NaPA on cell proliferation. PC3; DU145;LNCaP; and FS4 cultures were treated with NaPA or PAG for four days.

FIG. 12 shows a chromatogram of phenylacetate (PA) andphenylacetylglutamine (PAG). The peaks at 9.8 and 17.1 minutes representPAG and PA, respectively. Serum concentrations of 250 μg/ml in bothinstances.

FIG. 13 shows serum concentrations of PA ( ) and PAG ( ) and plasmaconcentrations of glutamine ( ) following a 150 mg/kg i.v. bolus of PAover 2 hours.

FIG. 14 shows declining phenylacetate concentrations over time duringCIVI (250 mg/kg/day) in one patient, suggestive of clearance induction.

FIG. 15 shows the inhibition of tumor cell invasion by NaPA cellstreated in culture for seven (7) days which were harvested and assayedfor their invasive properties using a modified Boyden Chamber with amatrigel-coated filter. Results were scored six (6) to twenty-four (24)hours later.

FIG. 16 shows a simulation of a q 12 hour PA regimen (200 mg/kg/dose, 1hour infusion) in a pharmacokinetically average patient. For simplicity,induction of clearance was not factored in.

FIG. 17 shows the effect of NaPA on cell growth and differentiation. (∘)Total cell number and (•) the fraction of benzidine-positive cells weredetermined after 4 days of continuous treatment. Data represent means±SD(n=4). Cell viability was greater than 95%.

FIGS. 18A and 18B show the time-dependent changes in cell proliferationand Hb production. NaPA (5 mM) was added on days 2, 4, 6, and 8 of phaseII cultures derived from normal donorsl, and the cells were analyzed onday 13. Panel 18A: Nubmer of Hb-containing cells per ml (×10⁻¹), and theamounts of Hb (pg) per cell (MCH). Panel 18B: Total Hb (pg) per mlculture, and the proportion of HbF out of total Hb (% HbF). Data pointsrepresent the means of four determinations. The deviation of results ofeach determination from the mean did not exceed 10%. NaPB at 2.5 mMproduced comparable effects (not shown). In all cases, cell viabilitywas over 95%.

FIG. 19 shows the effect of NaPA on the proportions of Hb species incultured erythroid precursors derived from a patient with sickle cellanemia. NaPA was added to 7 day phase II cultures. The cells wereharvested and lysed on day 13, and the proportions of HbF, HbA₂, and HbSwere determined following separation on cation exchange HPLC.

FIG. 20 shows the increased production of TGF-α by human keratinocytesupon treatment with NaPA and NaPB. Epithetial HK5 cells were treatedwith NaPB (3.0 mM, 1.5 mM, 0.75 mM), NaPB (10 mM, 5.0 mM, 2.5 mM) andPAG (5 mM) continuously for 4 days. Untreated cells served as a control.The amount of TGF-α (ng/ml/10⁶ cells) was measured by using anti-TGF-αantibodies.

FIG. 21 shows the enhanced expression of the surface antigens W6/32 (MHCclass I), DR (MHC class II) and ICAM-1 in melanoma cells treated withNaPB. Melanoma 1011 cells were treated with 2 mM PB for 10 days.Treatment was discontinued for 3 days to document the stability of theeffect. FACS analysis revealed markedly increased expression of theantigens following treatment (shaded area); the expression of thesurface antigens was similar or slightly greater on day 13 than on day10, indicating that PB induced terminal differentiation.

FIG. 22 shows the activation of the Peroxisomal Proliferator Receptor(PPAR) by PA, PB and various phenylacetic acid analogs. The activationis measured by the increased production of the indicator gene forcloramphenicol acetyl transferase (CAT), which is controlled by theresponse element for acyl-CoA oxidase, relative to the control (C). Theexperimental details this activation measurement method can be found inSher et al., Biochem., 32(21):5598 (1993)). The concentration (in mM) ofa particular drug is noted next to the following symbols for the variousdrugs: CF=clofibrate, PA=phenylacetate, CP=chlorophenylacetate,PB=phenylbutyrate, CPB=chlorophenylbutyrate, PAG=phenylacetylglutamine,IPB=iodophenylbutyrate, B=butyrate, IAA=indole acetic acid,NA=naphthylacetate, PP=phenoxypropionic acid, 2-4D=2,4-dichlorophenoxyacetate.

FIG. 23 shows the modulation by phenylbutyrate of glutathione (GSH),gamma-glutamyl transpeptidase (GGT) and catalase activites. Theantioxidant capacity (mM or units/mg protein) of the enzymes weremeasured for up to approximately 100 hours following treatment ofprostatic PC3 cells with 2 mM NaPB.

FIG. 24 shows the radiosensitization by PA and PB of human glioblastomaU87 cells by pretreatment for 72 hours with 1, 3 and 5 mM PA and 2.5 mMPB prior to exposure to ionizing radiation (Co⁶⁰ γ-radiaition).

FIG. 25 shows the inhibition of the growth of breast MCF-7adriamycin-resistant cancer cells by continuous exposure of up to 10 mMPA for 4 days.

FIG. 26 shows the relationship between lipophilicity and the cytostasisinduced by phenylacetate derivatives in prostate carcinoma cells and inplants. The log 1/IC₅₀ values for prostatic cells (calculated from datapresented in Table 21), were compared with the 1/IC₅₀ for rapidlydeveloping plant tissues. Tested compounds, listed in an increasingorder of their CLOGPs, included 4-hydroxy-PA, PA, 4-fluoro-PA,3-methyl-PA, 4-methyl-PA, 4-chloro-PA, 3-chloro-PA, and 4-iodo-PA.

FIG. 27 the phenotypic reversion induced by phenylacetate and selectedderivatives. The malignant prostatic PC3 cells were treated as describedin "Material and Methods". Data indicates the relative potency of testedcompounds in significantly inhibiting PC3 anchorage-independence 27(A)and completely blocking matrigel invasion 27(B). Phenylacetate andanalogs are presented in an increasing order of CLOGP (top to bottom).CLOGP values are provided in Tables 21 and 22. The effect onanchorage-dependency was confirmed with U87 cells (not shown).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "phenylacetic acid derivative" (or"phenylacetic acid analog") refers to a compound of the formula:##STR2## ; wherein R₀ is aryl (e.g., phenyl, napthyl), phenoxy,substituted aryl (e.g., one or more halogen [e.g., F, Cl, Br, I], loweralkyl [e.g., methyl, ethyl, propyl, butyl] or hydroxy substituents) orsubstituted phenoxy (e.g., one or more halogen [e.g., F, Cl, Br, I],lower alkyl [e.g., methyl, ethyl, propyl, butyl] or hydroxysubstituents);

R₁ and R₂ are each H, lower alkoxy (e.g., methoxy, ethoxy), lowerstraight and branched chain alkyl (e.g., methyl, ethyl, propyl, butyl)or halogen (e.g., F, Cl, Br, I);

R₃ and R₄ are each H, lower straight and branched chain alkyl (e.g.,methyl, ethyl, propyl, butyl), lower alkoxy (e.g., methoxy, ethoxy) orhalogen (e.g., F, Cl, Br, I); and

n is an integer from 0 to 2;

salts thereof (e.g., Na⁺, K⁺ or other pharmaceutically acceptablesalts); stereoisomers thereof; and mixtures thereof.

When n is equal to 2, each of the two R₃ substituents and each of thetwo R₄ substituents can vary independently within the above phenylaceticacid derivative definition. It is indended that this definition includesphenylacetic acid (PAA) and phenylbutyric acid (PBA). Mixtures accordingto this definition are intended to include mixtures of carboxylic acidsalts, for instance, a mixture of sodium phenylacetate and potassiumphenylacetate. Because the carboxylic portion of these compounds is theprimarily active portion, references herein to a carboxylate, such asphenylacetate (PA) or phenylbutyrate (PB), are intended to refer also toan appropriate counter cation, such as Na⁺, K⁺ or anotherpharmaceutically acceptable cation such as an organic cation (e.g.,arginine). Thus, as used herein, a PA or PB derivative or analog refersto the phenylacetic acid derivatives of this definition. Some of thesederivatives can be interconverted when present in a biological system.For instance, PA can be enzymatically converted to PB within an animaland, similarly, PB can be converted to PA.

Thus, phenylacetic acid derivatives include, without limitation,phenylacetic acid, phenylpropionic acid, phenylbutyric acid,1-naphthylacetic acid, phenoxyacetic acid, phenoxypropionic acid,phenoxybutyric acid, 4-chlorophenylacetic acid, 4-chlorophenylbutyricacid, 4-iodophenylacetic acid, 4-iodophenylbutyric acid,α-methylphenylacetic acid, α-methoxyphenylacetic acid,α-ethylphenylacetic acid, α-hydroxyphenylacetic acid,4-fluorophenylacetic acid, 4-fluorophenylbutyric acid,2-methylphenylacetic acid, 3-methylphenylacetic acid,4-methylphenylacetic acid, 3-chlorophenylacetic acid,3-chlorophenylbutyric acid, 2-chlorophenylacetic acid,2-chlorophenylbutyric acid and 2,6-dichlorophenylacetic acid, and thesodium salts of the these compounds.

The compounds of the present invention can be administeredintravenously, enterally, parenterally, intramuscularly, intranasally,subcutaneously, topically or orally. The dosage amounts are based on theeffective inhibitory concentrations observed in vitro and in vivo inantitumorigenicity studies. The varied and efficacious utility of thecompounds of the present invention is further illustrated by thefindings that they may also be administered concomitantly or incombination with other antitumor agents (such as hydroxyurea,5-azacytidine, 5-aza-2'-deoxycytidine, and suramin); retinoids;hormones; biological response modifiers (such as interferon andhematopoietic growth factors); and conventional chemo- and radiationtherapy or various combinations thereof.

The present invention also provides methods of inducing tumor celldifferentiation in a host comprising administering to the host atherapeutically effective amount of PAA or a pharmaceutically acceptablederivative thereof.

The present invention also provides methods of preventing the formationof malignancies by administering to a host a prophylactically effectiveamount of PAA or a pharmaceutically acceptable derivative thereof.

The present invention also provides methods of treating malignantconditions, such as prostatic cancer, melanoma, adult and pediatrictumors, e.g., brain tumors of glial origin, astrocytoma, Kaposi'ssarcoma, lung adenocarcinoma and leukemias, as well as hyperplasticlesions, e.g., benign hyperplastic prostate and papillomas byadministering a therapeutically effective amount of PAA or apharmaceutically acceptable derivative thereof.

In addition, the present invention provides methods of treatingconditions such as neuroblastoma, promyelocytic leukemia,myelodisplasia, glioma, prostate cancer, breast cancer, melanoma, andnon-small cell lung cancer.

It is understood that the methods and compositions of this invention canbe used to treat animal subjects, including human subjects.

According to the present invention, phenylacetic acid derivatives, andin particular NaPA and NaPB, have been found to be excellent inhibitorsof the growth of specific tumor cells, affecting the proliferation ofthe malignant cells while sparing normal tissues. Also, according to thepresent invention, NaPA and its analogs have been found to induce tumorcell differentiation, thus offering a very desirable approach to cancerprevention and therapy. Additionally, NaPA and its analogs have beenfound to be of value for the treatment of viral indications such asAIDS. NaPA is also implicated in the treatment of severe beta-chainhemoglobinopathies. The exact mechanisms by which the compounds used inthe methods of this invention exert their effects are uncertain. Onepotential mechanism may involve depletion of plasma glutamine. Based onthe data reported herein, it is believed that glutamine depletion alonecannot explain the molecular and phenotypic changes observed in vitrofollowing exposure to NaPA. It will be understood, however, that thepresent invention is not to be limited by any theoretical basis for theobserved results.

EXAMPLES

The herein offered examples, including experiments, provide methods forillustrating, without any implied limitation, the practice of thisinvention focusing on phenylacetic acid and its derivatives directed toA. Cancer therapy and prevention; B. Treatment and prevention of AIDS;C. Induction of fetal hemoglobin synthesis in β-chainhemoglobinopathies; D. Use of phenylacetic acid and its derivatives inwound healing; E. Use of phenylacetic acid and its derivatives intreatment of diseases associated with interleukin-6; F. Use ofphenylacetic acid and its derivatives in the treatment ofAIDS-associated CNS dysfunction; G. Use of phenylacetic acid and itsderivatives to enhance immunosurveillance; H. Method of monitoring thedosage level of phenylacetic acid and its derivatives in a patientand/or the patient's response to these drugs; I. The activation of thePPAR by phenylacetic acid and its derivatives; J. Use of phenylaceticacid and its derivatives in treatment of cancers having a multiple-drugresistant phenotype; and K. phenylacetic acid and its derivatives,correlation between potency and lipophilicity.

SECTION A: PHENYLACETATE IN CANCER PREVENTION AND MAINTENANCE THERAPY

Recent advances in molecular techniques enable the detection of geneticdisorders associated with a predisposition to cancer. Consequently, itis now possible to identify high-risk individuals as well as patients ina state of remission but afflicted with a residual disease. Despite suchremarkable capabilities, there is still no acceptable preventivetreatment. Chemopreventive drugs are also needed for adjuvant therapy,to minimize the carcinogenic effects of the prevailing anticancer agentsand yet maintain tumor responses.

To qualify for use in chemoprevention, a potential drug should haveantitumor activities, be non-toxic and well tolerated by humans, easy toadminister (e.g., orally or intravenously), and inexpensive. We suggestthat NaPA possesses all of the above characteristics.

1. Prevention of Neoplastic Transformation--Oncogene Transfer Studies

NIH 3T3 cells carrying activated Ejras oncogene (originally isolatedfrom human bladder carcinoma) were used as a model to study thepotential benefit of NaPA treatment to high risk individuals, in whompredisposition is associated with oncogene activation. Cell treatmentwith NaPA was initiated 24-48 hours after oncogene transfer. Results,scored 14-21 days later, show dose-dependent reduction in the formationof ras-transformed foci in cultures treated with NaPA. Molecularanalyses indicated that the drug did not interfere with oncogene uptakeand transcription, but rather prevented the process of neoplastictransformation. The effect was reversible upon cessation of treatment.In treated humans, however, the fate of the premalignant cells may besubstantially different due to involvement of humoral and cellularimmunity (see discussion below).

2. Prevention of tumor progression by genotoxic chemotherapy

Current approaches to combat cancer rely primarily on the use ofchemicals and radiation, which are themselves carcinogenic and maypromote recurrences and the development of metastatic disease. Oneexample is the chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC).While this drug shows promise in treatment of some leukemias and severeinborn anemias, the clinical applications have been hindered by concernsregarding toxicity and carcinogenic effects. However, for the first timethe data indicate that NaPA can prevent tumor progression induced bytreatment with 5AzadC.

The experimental model involved nonmalignant 4C8a10 cells (revertants ofHa-ras-transformed NIH 3T3 fibroblasts). Transient treatment of thepremalignant cells with 5AzadC resulted in malignant conversion evidentwithin 2 days, as determined by cell morphology, loss of contactinhibition and anchorage dependent growth in culture, and acquiredinvasive properties and tumorigenicity in recipient athymic mice.Remarkably, NaPA prevented the development of these malignant phenotypesin the 5AzadC treated cultures (Table 1).

                  TABLE 1                                                         ______________________________________                                                    Tumor Formation.sup.a                                                                       Growth                                              Treatment    Incidence  Size (mm) on matrigel.sup.b                           ______________________________________                                        None         3/8        1 (0.5-2) -                                           5AzadC (0.1 uM)                                                                            8/8        11.5 (4-19)                                                                             +                                           NaPA (1.5 mg/ml)                                                                           0/8                  -                                           5AzadC + NaPA                                                                              0/8        0         -                                           (0.1 uM) (1.5 mg/ml)                                                          ______________________________________                                         .sup.a Cells pretreated in culture were injected s.c. (5 × 10.sup.5     cells per site) into 3 month old female athymic nude mice (Division of        Cancer Treatment, NCI Animal Program, Frederick Cancer Research Facility)     Results indicate the incidence (tumor bearing/injected animals), as well      as tumor size as mean (range), determined after 3 weeks.                      .sup.b Cells were plated on top of matrigel (reconstituted basement           membrane) and observed for malignant growth pattern, i.e., active             replication, development of characteristic processes, and invasion.      

3. Activity in Humans.

In terms of cancer prevention, the beneficial effect of NaPA to humansmay be even more dramatic than that observed with the experimentalmodels. In humans, NaPA is known to deplete circulating glutamine, anamino acid critical for the development and progression of cancer. Theenzymatic reaction leading to glutamine depletion takes place in theliver and kidney. It is not clear whether or not glutamine depletionoccurs in the cultured tumor cells. Moreover, molecular analysisrevealed that NaPA induced the expression of histocompatibility class Iantigens, which are localized on the surface of tumor cells and affectthe immune responses of the host. While the therapeutic benefit of NaPAobserved in cultures is in some cases reversible upon cessation oftreatment, in patients the residual tumor cells would eventually beeliminated by the immune system. Even if chemoprevention will requirecontinuous treatment with NaPA, such treatment would be acceptableconsidering the lack of toxicity.

Pharmaceutical compositions containing phenylacetate have been shown tocause reversal of malignancy and to induce differentiation of tumorcells. To demonstrate the capacity of drugs to induce differentiation oftumor cells, three in vitro differentiation model systems and one invivo phase I clinical trial were used (further described herein). Thefirst system used a human promyelocytic leukemia cell line HL-60. Thiscell line represents uncommitted precursor cells that can be induced toterminally differentiate along the myeloid or monocytic lineage. In thesecond system, immortalized embryonic mesenchymal C3H 10T1/2 cells wereused which have the capability of differentiating into myocytes,adipocytes, or chondrocytes. In the third system, human erythroleukemiaK562 cells were used because they can be induced to produce hemoglobin.Finally, the in vivo experiments demonstrated the efficacy of NaPA ininducing terminal differentiation in humans and animals.

NaPA and NaPB have also been shown to affect tumor growth in vitro andin animal models at pharmacological, non-toxic concentrations. Thesearomatic fatty acids induced cytostasis and promoted maturation ofvarious human malignant cells, including hormone-refractory prostaticcarcinoma, glioblastoma, malignant melanoma, and lung carcinoma. Themarked changes in tumor biologoy were associated with alterations in theexpression of genes implicated in tumor growth, invasion, anglogenesis,and immunogenicity. Multiple mechansims of drug action appear to beinvolved. These mechanisms include (a) modification of lipid metabolism,(b) regulation of gene expression through DNA hypomethylation andtranscriptional activation, and (c) inhibition of proteinisoprenylation. Phase I clinical trials confirmed the efficacy of thesenovel, nontoxic differentiation inducers (see Example 15).

EXAMPLE 1

HL-60 and 10T1/2 cells--PAG and NaPA treatment

Referring now to the data obtained using the first system (resultsillustrated in FIG. 1), logarithmically growing HL-60 [--•--] and 10T1/2[--∘--] cells were treated for four days with NaPA [solid line] orphenylacetylglutamate (PAG) [dashed line]. The adherent cells weredetached with trypsin/EDTA and the cell number determined using ahemocytometer. Data points indicate the mean±S.D. of duplicates from twoindependent experiments. The cell lines were obtained from the AmericanType Culture Collection and maintained in RPMI 1640 (HL-60) orDulbecco's Modified Eagle's Medium (10T1/2) supplemented with 10% heatinactivated fetal calf serum (Gibco Laboratories), 2 mM L-Gtutamine, andantibiotics. PAA (Sigma, St. Louis Mo.) and PAG were each dissolved indistilled water, brought to pH 7.0 by the addition of NaOH, and storedin -20° C. until used. As demonstrated in FIG. 1, NaPA treatment of theHL-60 and 10T1/2 cultures was associated with dose dependent inhibitionof cell proliferation.

EXAMPLE 2

HL-60 cells--induction of granulocyte differentiation

To further evaluate the effectiveness of NaPA as an inducer of tumorcell differentiation, the ability of NaPA to induce granulocytedifferentiation in HL-60 was investigated. The ability of cells toreduce nitroblue tetrazolium (NBT) is indicative of oxidase activitycharacteristic of the more mature forms of human bone marrowgranulocytes. NBT reduction thus serves as an indicator of granulocytedifferentiation. In FIG. 2, the number of NBT positive cells wasdetermined after 4 days [solid bars] or 7 days [hatched bar] oftreatment. NaPA (h), 1.6 mg/ml; NaPA (1), 0.8 mg/ml.4-hydroxyphenylacetate (4HPA) and PAG were used at 1.6 mg/ml.Potentiation by retinoic acid (RA) 10 nM was comparable to that byinterferon gamma 300 IU/ml. The direction of differentiation towardsgranulocytes in cultures treated with NaPA, whether used alone or incombination with RA, was confirmed by microscopic evaluation of cellsstained with Wright Stain and the lack of nonspecific esterase activity.The effect of acivicin (ACV) 1 μg/ml was similar to6-diazo-5-oxo-L-norleucine (DON) 25 μg/ml. Glutamine starvation (Gln,<0.06 mM) was as described. Cell viability determined by trypan blueexclusion was over 95% in all cases, except for DON and ACV which were75% and 63%, respectively. DON, ACV and HPA are glutamine antagonists.As illustrated in FIG. 2, it is clear that NaPA is capable of inducinggranulocyte differentiation in HL-60. As further illustrated in FIG. 2,differentiation of HL-60, assessed morphologically and functionally, wassequential and could be further enhanced by the addition of low doses ofretinoic acid [RA, 10 nM) or interferon gamma (300 IU/ml). After sevendays of NaPA treatment, or four days, when combined with RA, the HL-60cultures were composed of early stage myelocytes and metamyelocytes(30-50%), as well as banded and segmented neutrophils (30-40%) capableof NBT.

Pharmacokinetics studies in children with urea cycle disorders indicatethat infusion of NaPA 300-500 mg/kg/day, a well tolerated treatment,results in plasma levels of approximately 800 μg/ml. [Brusilow, S. W. etal. Treatment of episodic hyperammonemia in children with inborn errorsof urea synthesis. The New England Journal of Medicine. 310:1630-1634(1984).] This same concentration was shown to effectively induce tumorcell differentiation in the present experimental system.

EXAMPLE 3

10T1/2 cells--NaPA induction of adipocyte conversion

FIG. 3 illustrates that NaPA is capable of inducing adipocyte conversionin 10T1/2 cultures. Confluent cultures were treated with NaPA for sevendays. Lower: Quantitation of adipocytosis. Cells were fixed with 37%formaldehyde and stained with Oil-Red O. The stained intracellular lipidwas extracted with butanol, and the optical density was determined usinga Titertek Multiskan MC, manufactured by Flow Laboratories, at awavelength 510 nm. Increased lipid accumulation was evident in cellstreated with as little as 0.024 mg/ml of NaPA. The results in FIG. 3show that differentiation was dose- and time-dependent, and apparentlyirreversible upon cessation of treatment. NaPA at 800 μg/ml wasefficient and totally free of cytotoxic effect. In the 10T1/2 model,adipoocyte conversion involved over 80% of the cell population. It wasnoted that higher drug concentrations further increased the efficiencyof differentiation as well as the size of lipid droplets in each cell.

It is known that glutamine conjugation by NaPA is limited to humans andhigher primates and that in rodents NaPA instead binds glycine. [James,M. O. et al. The conjugation of phenylacetic acid in man, sub-humanprimates and some non-primate species. Proc. R. Soc. Lond. B. 182:25-35(1972).] Consequently, the effect of NaPA on the mouse 10T1/2 cell linecould not be explained by an effect on glutamine. In agreement, neitherglutamine starvation nor treatment with glutamine antagonists such asDON and ACV resulted in adipocyte conversion.

EXAMPLE 4

Induction of lipid accumulation and adipocyte differentiation

4. Clinical use of phenylacetate and derivatives

                  TABLE 2                                                         ______________________________________                                        Phenylacetate and Derivatives: Induction of cellular                          differentiation in premalignant 10T1/2 cells                                  Compounds       Differentiation at 1 mM                                                                       DC.sub.50 *                                   (sodium salts)  (%)             (mM)                                          ______________________________________                                        Phenylacetate   65              0.7                                           1-naphthylacetate                                                                             >95             <0.1                                          3-chlorophenylacetate                                                                         80              0.5                                           4-chlorophenylacetate                                                                         50              1.0                                           2,6-dichlorophenylacetate                                                                     75              0.5                                           4-fluorophenylaceatae                                                                         65              0.7                                           ______________________________________                                         *DC.sub.50, concentration of compound causing 50% differentiation        

As shown in Table 2, phenylacetate and its derivatives efficientlyinduced lipid accumulation and adipocyte differentiation in premalignantcells. These and other results indicate that the tested compounds mightbe of value in:

A. Cancer prevention. Non-replicating, differentiated tumor cells arenot likely to progress to malignancy.

B. Differentiation therapy of malignant and pathological nonmalignantconditions.

C. Treatment of lipid disorders, in which patients would benefit fromincreased lipid accumulation.

D. Wound healing. This is indicated by the ability of phenylacetate toinduce collagen synthesis in fibroblasts (see Section D herein).

Studies in plants have revealed that NaPA can interact withintracellular regulatory proteins and modulate cellular. RNA levels. Inan attempt to explore the possible mechanism of action, Northern blotanalysis of HL-60 and 10T1/2 cells was performed according toconventional methods. Cytoplasmic RNA was extracted, separated andanalyzed (20 μg/lane) from confluent cultures treated for 72 hours withNaPA or PAG (mg/ml); C is the untreated control. The aP2 cDNA probe waslabeled with [³² P]dCTP (New England Nuclear) using a commerciallyavailable random primed DNA labeling kit. Ethidium bromide-stained 28SrRNA indicates the relative amounts of total RNA in each lane.

The results of the Northern blot analysis of HL-60 and 10T1/2 cells,showed marked changes in gene expression shortly after NaPA treatment.Expression of the adipocyte-specific aP2 gene was induced within 24hours in treated 10T1/2 confluent cultures reaching maximal mRNA levelsby 72 hours.

EXAMPLE 5

HL-60 cells--myc down regulation

In HL-60, cell transformation has been linked to amplification andover-expression, and differentiation would typically require downregulation of myc expression. [Collins, S. J. The HL-60 promyelocyticleukemia cell line: Proliferation, differentiation, and cellularoncogene expression. Blood. 70:1233-1244 (1987)]. To demonstrate thekinetics of myc inhibition and HLA-A induction, Northern blot analysisof cytoplasmic RNA (20 μg/lane) was carried out on cells treated withNaPA and PAG for specified durations of time and untreated controls (-).The dose-dependency and specificity of the effect of NaPA was observed.Two concentrations of NaPA, 1.6 mg/ml (++) and 0.8 mg/ml (+), and PAG at1.6 mg/ml were investigated. The ³² P-labeled probes used were myc 3rdexon (Oncor) and HLA-A3 Hind III/EcoRI fragment. NaPA caused a rapiddecline in the amounts of myc mRNA. This occurred within 4 hours oftreatment, preceding the phenotypic changes detectable by 48 hours,approximately two cell cycles, after treatment. Similar kinetics of mycinhibition have been reported for other differentiation agents such asdimethyl sulfoxide, sodium butyrate, bromodeoxyuridine, retinoids, and1,25-dihydroxyvitamin D₃. The results observed suggest that downregulation of oncogene expression by NaPA may be responsible in part forthe growth arrest and induction of terminal differentiation. Inaddition, it is evident that NaPA treatment of the leukemic cells wasassociated with time- and dose-dependent accumulation of HLA-A mRNAcoding for class I major histocompatibility antigens. This enhances theimmunogenicity of tumors in vivo.

EXAMPLE 6

K562 cells--NaPA promotes hemoglobin biosynthesis

Further support for the use of NaPA as a non-toxic inducer of tumor celldifferentiation is found in the ability of NaPA to promote hemoglobinbiosynthesis in erythroleukemia cells. K562 leukemic cells have anonfunctional beta-globin gene and, therefore, do not normally producesignificant amounts hemoglobin. When K562 human erythroleukemia cellswere grown in the presence of NaPA at 0.8 and 1.6 mg/ml concentrations,hemoglobin accumulation, a marker of differentiation, was found toincrease 4 to 9 fold over that of control cells grown in the absence ofNaPA. Hemoglobin accumulation was determined by Benzidine staining ofcells for hemoglobin and direct quantitation of the protein. The resultsof this study are reported in Table 16.

It has been shown that high concentrations of NaPA inhibit DNAmethylation in plants. [Vanjusin, B. J. et al. Biochemia 1, 46:47-53(1981)]. Alterations in DNA methylation can promote oncogenesis in theevolution of cells with metastatic capabilities. [Rimoldi, D. et al.Cancer Research. 51:1-7 (1991)]. These observations prompted someconcerns regarding potential long-term adverse effects with the use ofNaPA. To determine the potential tumorigenicity of NaPA, a comparativeanalysis was performed using NaPA and the known hypomethylating agent5-aza-2'-deoxycytidine (5AzadC).

Premalignant cells (3-4×10⁵) were plated in 75 cm² dishes and 5AzadC 0.1μM was added to the growth medium at 20 and 48 hrs after plating. Thecells were then subcultured in the absence of the nucleoside analog foran additional seven weeks. Cells treated with NaPA at 1.6 mg/ml weresubcultured in the continuous presence of the drug. For thetumorigenicity assay, 4-5 week-old female athymic nude mice wereinoculated s.c. with 1×10⁶ cells and observed for tumor growth at thesite of injection.

The results set forth in Table 3 show that NaPA, unlike the cytosineanalog, did not cause tumor progression.

                  TABLE 3                                                         ______________________________________                                        Tumorigenicity of C3H 10T1/2 Cells                                            in Athymic Mice                                                                        Tumors                                                                         Incidence                                                                     (positive/   Diameter   Time                                        Treatment injected mice)                                                                             (mm ± S.D.)                                                                           (weeks)                                     ______________________________________                                        None      0/8          0          13                                          5AzadC    8/8          5.5 ± 2.5                                                                              8                                          NaPA      0/8          0          13                                          ______________________________________                                    

The transient treatment of actively growing 10T1/2 cells with 5AzadCresulted in the development of foci of neoplastically transformed cellswith a frequency of about 7×10⁻⁴. These foci eventually became capableof tumor formation in athymic mice. By contrast, actively replicating10T1/2 cultures treated for seven weeks with NaPA, 800-1600 μg/ml,differentiated solely into adipocytes, forming neither neoplastic fociin vitro nor tumors in vivo in recipient mice.

Furthermore, experiments have demonstrated that NaPA can preventspontaneous or 5AzadC-induced neoplastic transformation, thusdemonstrating its novel role in cancer prevention. It is known that thetreatment of premalignant 4C8 and 10T1/2 cells with carcinogens such as5AzadC produces malignant conversion of the respective cells. When 4C8[Remold: et al., Cancer Research, 51:1-7 (1990)] and 10T1/2 cells wereexposed to 5AzadC, malignant conversion became evident in two days andtwo weeks, respectively. NaPA (0.8-1.6 mg/ml) prevented the appearanceof the malignant phenotype, as determined by cell morphology, contactinhibition and anchorage dependent growth in culture.

EXAMPLE 7

Growth arrest in malignant gliomas

In addition, Phenylacetate has been implicated in damage to immaturebrain in phenylketonuria. Because of similarities in growth pattern andmetabolism between the developing normal brain and malignant centralnervous system tumors, phenylacetate may be detrimental to some braincancers. Phenylacetate can induce cytostasis and reversal of malignantproperties of cultured human glioblastoma cells, when used atpharmacological concentrations that are well tolerated by children andadults. Interestingly, treated tumor cells exhibited biochemicalalterations similar to those observed in phenylketonuria-likeconditions, including selective decline in de novo cholesterol synthesisfrom mevalonate. Since gliomas, but not mature normal brain cells, arehighly dependent on mevalonate for production of sterols and isoprenoidsvital for cell growth, phenylacetate would be expected to affect tumorgrowth in vivo, while sparing normal tissues. Systemic treatment of ratsbearing intracranial gliomas resulted in significant tumor suppressionwith no apparent toxicity to the host. The experimental data, which areconsistent with clinical evidence for selective activity againstundifferentiated brain, suggest that phenylacetate may offer a safe andeffective novel approach to treatment of malignant gliomas.

Clinical experience, obtained during phenylacetate treatment of childrenwith urea cycle disorders, indicates that millimolar levels can beachieved without significant adverse effects. The lack of neurotoxicityin these patients is, however, in marked contrast to the severe braindamage documented in phenylketonuria (PKU), an inborn error ofphenylalanine metabolism associated with excessive production ofphenylacetate, microcephaly, and mental retardation. [Scriver, C. R.,and C. L. Clow. 1980. Phenylketonuria: epitome of human biochemicalgenetics. New Engl. J. Med. 303: 1394-1400.] The differences in clinicaloutcome can be explained by the fact that, although phenylacetatereadily crosses the blood-brain barrier in both prenatal and postnatallife, neurotoxicity is limited to the immature brain. Compellingevidence for a developmentally restricted window of susceptibility isprovided by the phenomenon of "maternal PKU syndrome": PKU females whoare diagnosed early and maintained on a phenylalanine-restricted diet,develop normally and subsequently tolerate a regular diet. These womenoften give birth to genetically normal, yet mentally retarded infantsdue to the untreated maternal PKU. The elevated levels of circulatingphenylacetate, while sparing the mature tissues of the mother, aredetrimental to the fetal brain. The primary pathological changes in PKUinvolve rapidly developing glial cells and are characterized byalterations in lipid metabolism and myelination with subsequent neuronaldysfunction. The vulnerable fetal glial tissues resemble neoplasticglial cells in numerous molecular and biochemical aspects, includingunique dependence upon mevalonate (MVA) metabolism for synthesis ofsterols and isoprenoids critical to cell replication [Kandutsch, A. A.,and S. E. Saucier. 1969. Regulation of sterol synthesis in developingbrains of normal and jimpy mice. Arch. Biochem. Biophys. 135: 201-208;Fumagalli, R., E. Grossi, P. Paoletti, and R. Paoletti. 1964. Studies onlipids in brain tumors. I. Occurrence and significance of sterolprecursors of cholesterol in human brain tumors. J. Neurochem. 11:561-565; Grossi, E., P. Paoletti, and R. Paoletti. 1958. An analysis ofbrain cholesterol and fatty acid biosynthesis. Arch. Int. Physiol.Biochem. 66: 564-572], and on circulating glutamine as the nitrogendonor for DNA, RNA and protein synthesis [Perry, T. L., S. Hasen, B.Tischler, R. Bunting, and S. Diamond. 1970. Glutamine depletion inphenylketonuria, a possible cause of the mental defect. New Engl. J.Med. 282: 761-766; Weber, G. 1983. Biochemical strategy of cancer cellsand the design of chemotherapy: G. H. A. Clowes Memorial Lecture. CancerRes. 43: 3466-3492]. The hypothesis underlying these studies was thatphenylacetate, known to conjugate and deplete serum glutamine in humans,and to inhibit the MVA pathway in immature brain [Castillo, M., M. F.Zafra, and E. Garcia-Peregrin. 1988. Inhibition of brain and liver3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphatedecarboxylase in experimental hyperphenylalaninemia. Neurochem. Res. 13:551-555; Castillo, M., J. Iglesias, M. F. Zafra, and E. Garcia-Peregrin.1991. Inhibition of chick brain cholesterogenic enzymes by phenyl andphenolic derivatives of phenylalanine. Neurochem. Int. 18: 171-174;Castillo, M., M. Martinez-Cayuela, M. F. Zafra, and E. Garcia-Peregrin.1991. Effect of phenylalanine derivatives on the main regulatory enzymesof hepatic cholestrogenesis. Mol. Cell. Biochem. 105: 21-25], mightattack these critical control points in malignant gliomas. The efficacyof phenylacetate was demonstrated using both in vitro and vivo tumormodels.

Cell Cultures and Reagents.

Human glioblastoma cell lines were purchased from the American TypeCulture Collection (ATCC, Rockville, Md.), and maintained in RPMI 1640supplemented with 10% heat inactivated fetal calf serum, antibiotics and2 mM L-glutamine, unless otherwise specified. Human umbilical veinendothelial cells, isolated from freshly obtained cords, were providedby D. Grant and H. Kleinman (NIH, Bethesda Md.). Sodium salts ofphenylacetic acid and of phenylbutyric acid were provided by ElanPharmaceutical Corporation (Gainseville, Ga.). Phenylacetylglutamine wasa gift from S. Brusilow (Johns Hopkins, Md.).

Evaluation of Cell Replication and Viability. Growth rates weredetermined by an enzymatic assay using3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertrazolium bromide (Sigma,St. Louis, Mo.) [Alley, M. C., D. A. Scudiero, A. Monks, M. L. Hursey,M. J. Czerwinski, D. L. Fine, B. J. Abbott, J. G. Mayo, R. H.Schoemaker, and M. R. Boyd. 1988. Feasibility of drug screening withpanels of human tumor cell lines using a microculture tetrazolium assay.Cancer Res. 48: 589-601], cell enumeration with a hemocytometerfollowing detachment with trypsin/EDTA, and by thymidine incorporationinto DNA. The different assays produced essentially the same results.Cell viability was assessed by trypan blue exclusion.

Colony Formation in Semi-Solid Agar.

Tumor cells were detached with trypsin/EDTA, re-suspended in growthmedium containing 0.36% agar, and placed onto a base layer of solid agar(0.9%) in the presence or absence of drugs. Colonies composed of 30 ormore cells were scored after three weeks.

Immunocytochemistry.

Cells were immunostained with anti-vimentin monoclonal antibodies usingDako PAP kit K537 (Dako Corporation, California).

Measurement of Cholesterol, Protein and DNA Synthesis.

For studies of steroid synthesis, cells were labeled for 24 hours with5×10⁶ DPM [5-3H]-mevalonate (35 Ci/mmol) (New England Nuclear, Boston,Mass.) in growth medium containing 3 μM lovastatin and 0.5 mM unlabeledmevalonate, in the presence or absence of 5 mM phenylacetate or 2.5 mMphenylbutyrate. Cellular steroids were extracted with hexane andseparated by silica thin layer chromatography. The R_(f) of thehexane-soluble radiolabled product was identical to that of aradiolabled cholesterol standard in three different solvent systems.Similarly treated cells were tested for de novo protein and DNAsynthesis by metabolic labeling with [³ H]-leucine (158 Ci/mmol) or [³H]-deoxythymidine (6.7 Ci/mmol) (New England Nuclear). Measurements of¹⁴ CO₂ released from [1-¹⁴ C]-mevalonate (49.5 mCi/mmol)(Amersham,Chicago, Ill.) in cell homogenates incubated withphenylacetate/phenylbutyrate were performed with minor modifications toestablished procedures.

Analysis of Protein Isoprenylation.

Cell cultures were incubated with 10 mM phenylacetate or 2.5 mMphenylbutyrate for 24 hours in complete growth medium, and labeled withRS-[2-¹⁴ C]-mevalonate (16 μCi/ml, specific activity 15 μCi/mmol)(American Radiolabeled Chemicals, Inc. St. Louis, Mo.) during the final15 hours of treatment. Whole cell proteins were extracted, resolved on10% SDS-polyacrylamide gels, and stained with Commassie Brilliant Blue.Gels were then dried and exposed to Kodak X-Omat film for 4 days.

Animal Studies.

To determine the effect of phenylacetate on the tumorigenic phenotype ofhuman glioblastoma cells, cultures were pre-treated for one week andthen harvested, resuspended in medium containing 30% matrigel(Collaborative Biomedical Products, Bedford, Mass.), and transplanteds.c. (2.5×10⁶ cells per site) into 5-week old female athymic mice(Division of Cancer Treatment, NCI Animal Program, Frederick CancerResearch Facility The animals were then observed for tumor growth at thesite of injection. To further evaluate drug efficacy in vivo, Fisher 344rats received a stereotaxic inoculation of syngeneic 9L gliosarcomacells (4×10⁴) into the deep white matter of the right cerebralhemisphere, as previously described [Weizsaecker, M., D. F., Deen, M. L.Rosenblum, T. Hoshino, P. H. Gutin, and M. Baker. 1981. The 9L rat braintumor: description and application of an animal model. J. Neurol. 224:183-192; Culver, K. W., Z. Ram, S. Walbridge, H. Ishii, E. H. Oldfield,and R. M. Blaese. 1992. In vivo gene transfer with retrovital vectorproducer cells for treatment of experimental brain tumors. Science. 256:1550-1552]. The animals were then subjected to two weeks of continuoustreatment with sodium phenylacetate (550 mg/kg/day, s.c.), using osmoticminipumps transplanted subcutaneously. In control rats the minipumpswere filled with saline. Statistical analysis of data employed theFisher's Exact Test.

Induction of cytostasis and phenotypic reversion in cultured humanglioblastoma cells. Treatment of glioblastoma cells with phenylacetateresulted in time- and dose-dependent growth arrest (FIG. 4), accompaniedby similarly diminished DNA synthesis. After 4-6 days of continuoustreatment with 4 mM phenylacetate, there was approximately 50%inhibition of growth in U87, A172, U373, U343, and HS683 cultures (IC₅₀4.4±0.6 mM). Reflecting on the heterogenous nature of tumor cellresponses, glioblastoma U251 and U138 cells were less sensitive withIC₅₀ values of 8-10 mM. Further studies, mimicking pharmacologicalconditions that are expected in patients, involved exposure of cells tophenylacetate in glutamine-depleted medium. These conditions completelyblocked glioblastoma cell growth, but had little effect on thereplication of normal endothelial cells (FIG. 5). Phenylbutyrate, anintermediate metabolite of phenylacetate formed in the brain by fattyacid elongation, also inhibited tumor cell replication (IC₅₀ 2.2±0.2 mMin A172, U87 and U373), while the end metabolite, phenylacetylglutamine,was inactive. In addition to inducing selective tumor cytostasis, bothphenylacetate and phenylbutyrate promoted cell maturation and reversionto a nonmalignant phenotype, manifested by an altered pattern ofcytoskeletal intermediate filaments, loss of anchorage-independence, andreduced tumorigenicity in athymic mice (Table 4). Immunocytochemicalanalysis of vimentin in phenylacetate-treated human glioblastoma U87cells showed altered morphology and cytoskeletal filament pattern. Thesechanges, confirmed by immunolabeling for glial fibrillary acidic proteinare consistent with cell maturation and correlate with reducedproliferative capacity and regained contact inhibition of growth. Theseprofound changes in tumor behavior were accompanied by alterations inthe expression of genes implicated in growth control, angiogenesis, andimmunosuppression (e.g., TGFα, HbF, and TGF-β2).

                  TABLE 4                                                         ______________________________________                                        Reversal of Malignancy of                                                     Human Glioblastoma Cells                                                                   Clonogenicity                                                                            Tumor Incidence.sup.2                                              in Soft Agar.sup.1                                                                       Positive/Injected                                     Treatment    (%)        Sites                                                 ______________________________________                                        None         8.1        9/10                                                  Phenylacetate                                                                 2.5 mM       0.5        ND                                                    5 mM         >0.01      2/10                                                  Phenylbutyrate                                                                1.25 mM      0.15       ND                                                    2.5 mM       >0.01      1/10                                                  ______________________________________                                         .sup.1 U87 cells were detached with trypsin/EDTA, resuspended in growth       medium containing 0.36% agar, and placed onto a base layer of solid agar      (0.9%) in the presence or absence of drugs. Colonies composed of 30 or        more cells were scored after three weeks.                                     .sup.2 U87cells pretreated in culture for one week, were harvested,           resuspended in medium containing 30% matrigel, and transplanted s.c. into     5week old female athymic mice (2.5 × 10.sup.6 cells per mouse). Dat     were recorded 5 weeks after cell inoculation.                                 ND = not determined.                                                     

Phenylacetate inhibits the mevalonate pathway and proteinisoprenylation. The most consistent biochemical change observed in glialcells exposed to phenylacetate involved alterations in lipid metabolismand inhibition of the MVA pathway (FIG. 6). Active de novo synthesis ofcholesterol and isoprenoids from precursors such as acetyl-CoA and MVAis an important feature of the developing brain (but not the maturebrain), coinciding with myelination. It is also a hallmark of malignantgliomas [Azarnoff, D. L., G. L. Curran, and W. P. Williamson. 1958.Incorporation of acetate-1-¹⁴ C into cholesterol by human intracranialtumors in vitro. J. Nat. Cancer Inst. 21: 1109-1115; Rudling, M. J., B.Angelin, C. O. Peterson, and V. P. Collins. 1990. Low densitylipoprotein receptor activity in human intracranial tumors and itsrelation to cholesterol requirement. Cancer Res. 50 (suppl): 483-487].Cholesterol production and protein isoprenylation diminished within 24hours of glioblastoma treatment with either phenylacetate orphenylbutyrate (FIG. 7), preceding changes in DNA and total proteinsynthesis, which were detectable after 48 hours. The reduction inisoprenylation was paralleled by a decrease in MVA decarboxylation (toless than 50% of control), an effect previously observed in embryonicbrain in PKU-like conditions. MVA-5-pyrophosphate decarboxylase, a keyenzyme regulating cholesterol synthesis in brain, is inhibited byphenylacetate under conditions in which MVA kinase and MVA-5-phosphatekinase are only minimally affected. Phenylacetate might also interferewith MVA synthesis from acetyl-CoA. Glioblastoma cells could not,however, be rescued by exogenous MVA (0.3-3 mM), suggesting that MVAutilization, rather than its synthesis, is the prime target. The declinein MVA decarboxylation and protein isoprenylation inphenylacetate-treated cells could be mimicked by using 1-2.5 mMphenylbutyrate.

Mevalonate is a precursor of several isopentenyl moieties required forprogression through the cell cycle such as sterols, dolichol, the sidechains of ubiquinone and isopentenyladenine, and prenyl groups thatmodify a small set of critical proteins [Goldstein, J. L., and M. S.Brown. 1990. Regulation of the mevalonate pathway. Nature. 343: 425-430;Marshall, C. J. 1993. Protein prenylation: A mediator of protein-proteininteractions. Science. 259: 1865-1866; Braun, P. E., D. De Angelis, W.W. Shtybel, and L. Bernier. 1991. Isoprenoid modification permits2',3'-cyclic nucleotide 3'-phosphodiesterase to bind to membranes. J.Neurosci. Res. 30: 540-544]. The latter include plasma membrane G andG-like proteins (e.g., ras) involved in mitogenic signal transduction(molecular weight 20-26 kDa), the myelination-related enzyme2',3'-cyclic nucleotide 3'-phosphodiesterase, and nuclear envelopelamins that play a key role in mitosis (44-74 kDa). Inhibition of steroland isoprenoid synthesis during rapid development of the brain couldlead to the microcephaly and impaired myelination seen in untreated PKU.Targeting MVA in dedifferentiated malignant gliomas, on the other hand,would be expected to inhibit tumor growth in vivo without damaging thesurrounding normal tissues, as the MVA pathway is significantly lessactive in mature brain.

Activity of phenylacetate in experimental gliomas in rats. To evaluatethe in vivo antitumor effect of phenylacetate, Fisher rats wereinoculated with stereotaxic intracerebral injection of syngeneic 9Lgliosarcoma cells. This tumor model is known for its aggressive growthpattern that results in nearly 100% mortality of rats within 3 to 4weeks. Phenylacetate was continuously administered by implantedsubcutaneous osmotic minipumps to deliver a clinically-achievable doseof 550 mg/kg/day. Systemic treatment for two weeks of rats bearingintracranial glioma cells markedly suppressed tumor growth (p<0.05,Table 5) with no detectable adverse effects. Further studies inexperimental animals indicate that phenylacetate (plasma andcerebrospinal fluid levels of 2-3 mM) induces tumor cell maturation invivo and significantly prolongs survival.

                  TABLE 5                                                         ______________________________________                                        Phenylacetate Activity in                                                     Experimental Brain Cancer                                                                     Brain Tumors.sup.2                                                        No.       Macro-    Micro-                                                                              Tumor                                   Treatment.sup.1                                                                           of animals                                                                              scopic    scopic                                                                              Free                                    ______________________________________                                        Saline      10        8         1     1                                       Phenylacetate                                                                             15        3         4     8                                       ______________________________________                                         .sup.1 Fisher 344 rats received a stereotaxic inoculation of syngeneic 9L     gliosarcoma cells into the deep white matter of the right cerebral            hemisphere, as described in Material and Methods. Animals were then           subjected to two weeks of continuous treatment with either sodium             phenylacetate (550 mg/kg/day, s.c.) or saline, using osmotic minipumps        transplanted subcutaneously.                                                  .sup.2 Animals were sacrificed 23 days after tumor inoculation to             determine antitumor effects. Findings were confirmed by histological          evaluation of the inoculated site.                                       

Summary and Prospective.

Phenylacetate has long been implicated in damage to the developing fetalbrain. As primary CNS tumors are highly reminiscent of immature fetalbrain, malignant gliomas should be equally vulnerable. Moreover, viewingmaternal PKU syndrome as a natural human model, phenylacetate would beexpected to suppress the growth of brain neoplasms without harmingnormal tissues. Experimental data supports this hypothesis.Phenylacetate induced selective cytostasis and promoted maturation ofglioma cells in vitro and in vivo. Premature growth arrest anddifferentiation could also underlie the damage to fetal brain in PKU.Multiple mechanisms of action are involved, including inhibition ofprotein isoprenylation and depletion of plasma glutamine in humans. Thedemonstrable antitumor activity, lack of toxicity, and ease ofadministration (oral or intravenous), demonstrate the clinical efficacyof phenylacetate in management of malignant gliomas, and perhaps ofother neoplasms as well. Previously, phenylacetate showed activity inprostate cancer in vitro. Phase I clinical studies with phenylacetate inthe treatment of adults with cancer confirmed that therapeutic levelscan be achieved in the plasma and cerebrospinal fluid with nosignificant toxicities, and provide preliminary evidence for benefit toprostatic carcinoma and glioblastoma patients (see Example 18).

Phenylacetate was used to treat human solid tumors, including prostaticcarcinoma, glioblastomas, and malignant melenoma. Treatment resulted inselective cytostasis and phenotypic reversion, as indicated by therestored anchorage-dependence, reduced invasiveness and loss oftumorigenicity in athymic mice. Molecular analysis of brain andhormone-refractory prostate cancer cells revealed marked decline in theproduction and secretion of TGFβ, a protein implicated in growthcontrol, angiogenesis, and immunosuppression. Treated prostatic cellsexhibited decreased proteolytic activity mediated byurokinase-plasminogen activator, a molecular marker of diseaseprogression in man.

EXAMPLE 8

Growth arrest, tumor maturation, and extended survival in brain tumorstreated with NaPA

In Vitro Studies.

Cell proliferation.

The effect of NaPA on cell proliferation was evaluated usingtritiatedthymidine incorporation assay on cultured 9L gliosarcoma cellsand cell enumeration using a hemocytometer following detachment withtrypsin/EDTA. 9L is a syngeneic malignant glial tumor derived fromFischer 344 rats and is associated with 100% mortality within three tofour weeks after intracerebral inoculation [Weizsaecker M, Deen D. F.,Rosenblum M. L., et al. The 9L rat brain tumor: description andapplication of an animal model. J Neuol. 1981; 224: 183-192]. Tumorcells were plated at 5×10⁴ tumor cells/well in 24-well plates (Costar,Cambridge, Mass.) in Dulbecco Modified Eagle's medium (DMEM) with 10%fetal bovine serum (Hyclone Laboratories Inc., Logan, Utah), 2 mML-glutamine (GIBCO BRL, Gaithersburg, Md.), 50 U/ml penicillin (GIBCO)and 50 μg/ml streptomycin (GIBCO) and 2.5 μg/ml Fungizone (ICNBiomedicals Inc., Costa Mesa, Calif.). After 24 hours, the medium waschanged and NaPA (Elan Pharmaceutical Research Corp., Gainesville, Ga.)added to the medium at 0, 2.5, 5, and 10 mM concentration for 5 days.Six hours before harvest, 0.5 mCi tritiatedthymidine (ICNRadiochemicals, Irvine, Calif.) was added to each well. Thymidineincorporation was determined by scintillation counting in triplicates.

Colony formation in semi-solid agar.

Anchorage independent growth (the ability of cells to form colonies insemi-solid agar) is characteristic of malignant glial cells. 9L cellswere harvested with trypsin/EDTA and resuspended at 1.0×10⁴ cells/ml ingrowth medium containing 0.36% agar (Difco). Two ml of the cellsuspension was added to 60 mm plates (Costar, Cambridge, Mass.) whichwere precoated with 4 ml of solid agar (0.9%). Phenylacetate was addedto the agar at different concentrations (0, 1.25, 2.5, and 5 mM). In asecond experiment, 9L cells were grown for 7 days in tissue culturecontaining 5 mM NaPA. The cells were then transferred, as described, toagar plates without NaPA. Colonies composed of 30 or more cells werecounted after 3 weeks.

9L brain tumor inoculation and phenylacetate administration.

Fisher 344 rats (n=50) weighing 230-350 grams were anesthetized usingintraperitoneal (i.p.) Ketamine (90 mg/Kg, Fort Dodge Laboratories,Inc., Fort Dodge, Iowa) and Xylazine (10 mg/Kg, Mobay Corporation,Shawnee, Kans.) and placed in a steriotaxic apparatus (David KopfInstruments, Tujunga, Calif.). 4×10⁴ syngeneic 9L gliosarcoma cells in 5μL (Hank's) balanced salt solution were injected into the deep whitematter (depth of inoculation -3.5 mm) of the right cerebral hemisphereusing a 10 μL Hamilton syringe connected to the manipulating arm of thesterotaxic apparatus. In 10 rats, phenylacetate was administered bycontinuous subcutaneous (s.c.) release of the drug using two 2ML2osmotic pumps release rate of 5 μl/hr for 14 days (Alza Corporation,Palo Alto, Calif.). On the day of tumor inoculation the pumps wereimplanted in the subcutaneous tissue of both flanks. The concentrationof the drug in the pumps was 650 mg/ml (total of 2600 mg for both pumps)for a daily dose of 550 mg/kg per rat. The minipumps were replaced after14 days for a total treatment of 28 days. Fifteen additional ratsreceived NaPA, as described, starting 7 days after intracerebralinoculation of the tumor. In these rats, an additional daily injectionof NaPA (300 mg/kg, i.p.) was given for 28 days. Control rats (n=25)received continuous saline from two s.c. 2ML2 osmotic pumps.Perioperative penicillin (100,000 u/kg, i.m.) was given to all ratsbefore implantation of the minipumps. Survival was recorded in eachgroup. Three rats treated for established tumors and two control ratswere sacrificed 7 days after initiation of NaPA (14 days after tumorinoculation). These were used for electron microscopic studies oftreated tumors, in vivo proliferation assays, and measurement of NaPAlevels in the serum and CSF. Peripheral organs (heart, lung, spleen,liver, kidney, bowel, adrenal, and gonads) were harvested and subjectedfor a routine histological examination. Brain specimens were sectionedand stained for routine hematoxylin and eosin (H&E) and myelin stains(Luxol-fast blue) for evidence of drug-related toxicity.

Electron microscopy.

Animals were sacrificed by intracardiac perfusion with 1%paraformaldehyde and 2.5% gluteraldehyde in 0.1M sodium cacodylatebuffer at pH 7.4. Two hours later the fixed brains were washed in bufferand sliced into 1 mm thick coronal sections. The areas containing tumorswere further dissected into 1 mm^(j) cubes, post-fixed with 2% osmiumtetroxide in 0.1M sodium cacodylate buffer for 2 hours, washed inbuffer, mordanted en block with 1% uranyl acetate at pH 5 overnight,then washed, dehydrated and embedded in Epon. Thin sections were cut atseveral levels into each block to ensure greater sampling. Electronmicrographs of tumor cells were taken at random for morphology.

In vivo proliferation assay.

One NaPA-treated and one saline-treated rat received an i.p. injectionof 9 mg/3 ml of BrdU (Amersham, Ill.) 14 days after tumor inoculationand 7 days after initiation of treatment. Two hours later the rats weresacrificed and the brains were removed and sectioned. Mouse anti-BrdUmonoclonal antibodies were used for immunostaining of the tissues whichwere then counterstained with hematoxylin. Tumor cells in 10 high-powerfields were enumerated in each tumor specimen and the percent ofpositively staining cells (indicating incorporation of BrdU duringactive cell division) was recorded.

Measurement of NaPA levels in serum and CSF.

Three NaPA-treated and 2 saline-treated rats were sacrificed after 7days of combined s.c. and i.p. NaPA or saline administration. Blood wasdrawn from the heart and CSF was aspirated from the cisterna magna. Dueto volume limitations of CSF, pooled serum and CSF samples were assessedin a similar fashion. Protein extraction of a 200 μl aliquot ofbiological fluid was carried out with 100 μl of a 10% perchloric acidsolution. 150 μl of supernate was neutralized with 25 μl of 20%potassium bicarbonate and centrifuged. 125 μl of supernate was thenpipetted into sampling tubes. Chromatography was performed on a Gilson715 HPLC system using a 30 cm Waters C18 column (i.d. 3.9 mm) at 60° C.A 75 μl injectate was eluted with an acetonitrile/water gradient rangingfrom 5 to 30% over 20 minutes and flowing at 1 ml/min. UV-monitoring wasperformed at a wavelength of 20 nm. Elution time for phenylacetate was14.8 minutes.

Statistical analysis.

The Chi-square test was used to compare proportions of BrdU-positivecells. The Mantel-Haenzel test was used to compare survival betweenNaPA-treated and saline-treated rats in the survival experiments.

In Vitro Results

In vitro Effect of NaPA on cell proliferation and anchorage dependency.Treatment of 9L cells with NaPA for 5 days resulted in dose-dependentdecrease in cell number with IC₅₀ at 6.0±0.5 mM. This was accompaniedwith a decrease in tritiated-thymidine incorporation (FIG. 8). Inaddition, phenylacetate induced a dose-dependent restoration ofanchorage dependency, indicating a reversion of the malignant phenotype(Table 19). 9L cells that were exposed to NaPA for 7 days before platingin agar (not containing NaPA) still showed >40% inhibition in colonyformation (Table 19).

                  TABLE 19                                                        ______________________________________                                        Phenylacetate Inhibits                                                        Anchorage-Independent Growth of 9L Gliosarcoma Cells                          Treatment PA in       Colony Formation                                        in Culture                                                                              Agar (mM)   # Colonies                                                                              % Inhibition                                  ______________________________________                                        none      0           628 ± 50                                                                             --                                            none      5            8 ± 4 98.7                                                    2.5         111 ± 13                                                                             82.4                                                    1.25        326 ± 20                                                                             48.0                                          .sup.a Phenylacetate                                                                    0           375 ± 25                                                                             40.3                                          ______________________________________                                         .sup.a 9L cells were treated with 5 mM phenylacetate in culture for 7 day     before being plated on soft agar.                                        

In Vivo Studies

In vivo proliferation assay and electron microscopy findings. Treatmentof established brain tumors with NaPA resulted in a significant decreasein the rate of proliferation. 285 of 1283 treated tumor cells stainedfor BrdU compared to 429 of 1347 saline-treated tumor cells (mitoticindex of 0.22 in NaPA-treated vs. 0.33 in saline-treated tumors;p<0.0001).

Electron microscopy of these tumors showed a striking abundance ofwell-organized rough endoplasmic reticulum in the NaPA-treated tumorcells, indicating a higher degree of cell differentiation [Ghadially FN.Endoplasmic reticulum and ribosomes in cell differentiation andneoplasia. In: eds. Ultrastructural Pathology of the Cell and Matrix.Third, London:Buttorworths; 1992:450-454]. By contrast, untreated tumorsgenerally had scant rough endoplasmic reticulum and numerouspolyribosomes, which are characteristics of highly malignant cells.

In addition, mitotic cells were more frequently found in untreatedtumors.

Serum and CSF levels of NaPA.

Assays of pooled serum and CSF from 3 treated and 2 control rats,obtained after 7 days of combined s.c. and i.p. NaPA (total daily doseof 850 mg/kg) or saline administration, revealed a mean phenylacetatelevel of 2.45 mM in the serum and 3.1 mM in the CSF. No phenylacetatewas detected in the serum of CSF samples from saline-treated rats.

Survival Experiments

Simultaneous tumor inoculation and administration of NaPA. Seven of 10NaPA-treated rats survived for >90 days after tumor inoculation whenNaPA was administered for 4 weeks starting on the day of tumorinoculation. Nine of 10 control rats died within 34 days after tumorinoculation (p<0.01, Mantel-Haenzel test) (FIG. 9).

Treatment of established tumors with NaPA.

Five of 12 rats treated with s.c. and i.p. NaPA for 4 weeks (starting 7days after tumor inoculation) are still alive 50 days after tumorinoculation, while 12 of 13 saline-treated rats died by day 36 (p<0.03,Mantel-Haenzel test) (FIG. 10).

Toxicity.

No adverse effects of NaPA treatment were detected in any treated rats.Histological evaluation of the major peripheral organs and non-tumoralbrain showed no abnormalities.

Discussion.

Phenylacetate induced a potent cytostatic and antitumor effect in the invitro and in vivo brain tumor models used in these studies. This effectextended beyond the duration of drug administration, indicated by thelong-term survival and apparent cure of rats which received NaPA eithersimultaneously with tumor inoculation or after tumors were established.This extended effect of NaPA shows that the malignant phenotype oftreated tumor cells reverted, perhaps irreversibly in some animals, toone that was more benign and differentiated. Anchorage independence,i.e., the ability of cells to form colonies in semi-solid agar, ischaracteristic of malignant glioma cells. Phenylacetate caused adose-dependent restoration of anchorage dependency, indicating reversionof the glioma cells to a non-malignant phenotype. More than 80%inhibition of colony formation was achieved at NaPA concentration in theagar plate of 2.5 mM, similar to the serum and CSF levels measured intreated rats. In addition, after one week of exposure to NaPA, more than40% of tumor cells maintained a benign growth pattern despite theabsence of NaPA in the agar plates (Table 19). A significant in vivoindicator of cell differentiation was observed in our study in thesubcellular organelles of treated brain-tumor cells. The disorganizedcytoplasmic polyribosomes in the saline-treated tumor cells weretransformed by NaPA to a hyperplastic, well organized, rough endoplasmicreticulum. The endoplasmic reticulum is a highly specialized structurethat performs many distinct functions. Hence a well-developedendoplasmic reticulum represents cell differentiation and functionalactivity. An inverse relationship has been noted between the amount ofrough endoplasmic reticulum and the growth rate and degree of malignancyof tumors [Ghadially FN. Diagnostic Electron Microscopy of Tumours. eds.2. London:Butterworth; 1985]. The numerous polyribosomes in theuntreated tumor cells correlated well with the number of mitoses seen bylight microscopy and were confirmed by the BrdU proliferation assay.These changes underscore the differentiating effect of NaPA on themalignant glial cells and correlate with the in vivo decrease in cellproliferation and extended survival that occurred in treated animalswith brain tumors.

Therapeutic blood and CSF NaPA levels were reached in the treated rats.The high CSF levels indicate good penetration of NaPA into the centralnervous system and into the developing tumor. The doses used are wellbelow the known toxic levels of NaPA in children with inborn errors ofurea synthesis (2.5 g/kg/d) or rats (1.6 g/kg/d) and indicate that NaPAcan be given safely at a higher doses, possibly with enhancement ofantitumor efficacy. These data indicate that phenylacetate, given torats at a non-toxic dose, has a profound effect on tumor growthregulation and cell maturation.

EXAMPLE 9

Suppression of 5-Aza-2'-deoxycytidine induced carcinogenesis

Differentiation inducers selected for their low cytotoxic and genotoxicpotential could be of major value in chemoprevention and maintenancetherapy. Specifically, the ability of phenylacetate to preventcarcinogenesis by the chemotherapeutic hypomethylating drug,5-aza-2'-deoxycytidine (5AzadC), was tested in vitro and in mice.Transient exposure of immortalized, but non-tumorigenic ras-transformed4C8 fibroblasts to 5AzadC resulted in neoplastic transformationmanifested by loss of contact inhibition of growth, acquiredinvasiveness, and tumorigenicity in athymic mice. The latter wasassociated with increased ras expression and a decline in collagenbiosynthesis. These profound phenotypic and molecular changes wereprevented by a simultaneous treatment with phenylacetate. Protectionfrom 5AzadC carcinogenesis by phenylacetate was: (a) highly efficientdespite DNA hypomethylation by both drugs; (b) free of cytotoxic andgenotoxic effects; (c) stable after treatment was discontinued, and; (d)reproducible in vivo. Whereas athymic mice bearing 4C8 cells developedfibrosarcomas following a single i.p. injection with 5AzadC, tumordevelopment was significantly inhibited by systemic treatment withnontoxic doses of phenylacetate. Phenylacetate and its precursorsuitable for oral administration, phenylbutyrate, may thus represent anew class of chemopreventive agents, the efficacy and safety of whichshould be further evaluated.

The multi-step nature of neoplastic transformation makes this diseaseprocess amendable to chemopreventive intervention. Several agents havebeen shown to inhibit carcinogenesis and thereby prevent the developmentof primary or secondary cancers [Kelloff, G. J., C. W. Boone, W. F.,Malone, and V. E. Steele. 1992. Chemoprevention clinical trials.Mutation Res., 267: 291-295; Weinstein, B. I. 1991. Cancer prevention:Recent progress and future opportunities. Cancer Res., 51:5080s-5085s;Wattenberg, L. W. Inhibition of carcinogenesis by naturally occurringand synthetic compounds. In: Y. Kuroda, D. M. Shankel and M. D. Waters(eds), Antimutagenesis and Anticarcinogenesis, Mechanisms II,pp.155-166. New York: Plenum Publishing Corp., 1990; Sporn, M. B., andD. L. Newton. 1979. Chemoprevention of cancer and retinoids. Fed. Proc.38:2528-2534]. Of major interest are natural products and their analogs,including vitamins (A, B12, C, D3, and E), retinoids, and terpenes.These agents can suppress neoplastic transformation subsequent to acarcinogenic insult by regulating cell growth and differentiation. Onesuch growth regulator is phenylacetate.

The efficacy of phenylacetate as a chemopreventive agent was testedusing in vitro and in vivo models of 5AzadC-induced carcinogenesis.Despite the promise of 5AzadC in the treatment of cancer and ofbeta-chain hemoglobinopathies, its clinical applications have beenhindered by concerns regarding carcinogenic potential. The model used inthe present studies involved premalignant murine fibroblasts (cell lines4C8 and PR4), which express a transcriptionally activated c-Ha-rasprotooncogene. These non-tumorigenic cells are highly susceptible tomalignant conversion by pharmacological doses of 5AzadC. However,Phenylacetate can protect such vulnerable cells from 5AzadC-inducedcarcinogenesis both in culture and in mice.

Cell Cultures and Reagents.

The subclones of mouse NIH 3T3 fibroblasts, PR4N and 4C8-A10 (designatedhere PR4 and 4C8) have been previously described [Wilson, V. L., R. A.Smith, H. Autrup, H. Krokan, D. E. Musci, N-N-T. Le, J. Longoria, D.Ziska, and C. C. Harris. 1986. Genomic 5-methylcytosine determination by³² P-postlabeling analysis. Anal. Biochem., 152:275-284; Dugaiczyk, A.,J. J. Haron, E. M. Ston, O. E. Dennison, K. N. Rothblum, and R. J.Schwartz. 1983. Cloning and sequencing of a deoxyribonucleic acid copyof glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acidisolated from chicken muscle. Biochem. 22:1605-1613]. Both cell linesare phenotypic revertants isolated from LTR/c-Ha-ras1-transformed 3T3cells after long-term treatment with murine interferon α/β. Cultureswere maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% heat inactivated fetal calf serum (Gibco) andantibiotics. The sodium salts of phenylacetic and phenylbutyric acids(Elan Pharmaceutical Corporation) were dissolved in distilled water.5AzadC (Sigma St. Louis Mo.) was dissolved in phosphate buffered saline(PBS) and stored in aliquots at -20° C. until use. Exposure of 5AzadC todirect light was avoided at all times to prevent drug hydrolysis.

Treatments with 5AzadC.

For treatment in culture, cells were plated at 1-2×10⁵ cells in 100 mmdishes and the drugs added to the growth medium at 20 and 48 hrs later.The cells were subsequently subcultured in the absence of the nucleosideanalogs and observed for phenotypic alterations. For in vivo treatmentwith 5AzadC, 6-9 week-old female athymic nude mice (Division of CancerTreatment, NCI Animal Program, Frederick Cancer Research Facility) wereinoculated subcutaneously (s.c.) with 0.5×10⁶ cells. Twenty four hourslater 400 μg of freshly prepared 5AzadC in 200 μl of PBS wasadministered intraperitoneally (i.p.) into each animal (approximately 20mg/kg). Systemic treatment with NaPA is described in the text.

Growth on Matrigel.

The ability of cells to degrade and cross tissue barriers was assessedby a qualitative vitro invasion assay that utilize matrigel, areconstituted basement membrane (Collaborative Research). Cells wereexposed for 48 hrs in T.C. plastic dishes with 5AzadC alone or incombination with NaPA. NaPA treatment continued for additional 1-2weeks. Cells were then replated (at 5×10⁴ per point) onto 16 mm dishes(Costar, Cambridge, Mass.), which were previously coated with 250 ofmatrigel (10 mg/ml). NaPA was either added to the dishes or omitted inorder to determine the reversibility of effect. Net-like formationcharacteristic of invasive cells occurred within 12 hours; invasion intothe matrigel was evident after 6-9 days.

Tumor Formation in Athymic Mice.

Cells were injected s.c. (5×10⁵ cells per site) into 4-6 week old femaleathymic nude mice (Division of Cancer Treatment, NCI animal Program,Frederick Cancer Research Facility). The number, size, and weight oftumors were recorded after 3-4 weeks. For histological examination,tumors were excised, fixed in Bouin's solution (picric acid: 37%formaldehyde: glacial acetic acid, 15:5:1 vol/vol), and stained withH&E.

Measurement of DNA Methylation.

To determine the 5-methylcytosine content, samples of cultures weretaken 24 hours after the second 5AzadC treatment. The cell pellets werelysed in 0.5% SDS, 0.1M NaCl, 10 mM EDTA pH 8.0, added with 400 μg/ml ofproteinase K (Boehringer Mannheim), and stored at -70° C. until DNAisolation and analysis. The content of methylated/unmethylated cytosineresidues in the cellular DNA was measured by a ³² P-postlabelingtechnique as previously described.

Northern Blot Analysis and DNA Probes.

Cytoplasmic RNA was extracted from exponentially growing cells andseparated by electrophoresis in 1.2% agarose-formaldehyde gels. RNApreparation, blotting onto nylon membranes (Schleicher and Schuell),hybridization with radiolabeled DNA probes, and autoradiography wereperformed as described [Rimoldi, D., V. Srikantan, V. L. Wilson, R. H.Bassin, and D. Samid. 1991. Increased sensitivity of nontumorigenicfibroblasts expressing ras or myc oncogenes to malignant transformationinduced by 5-aza-2'-deoxycytidine. Cancer Res., 51:324-330]. The DNAprobes included: 6.2 kb EcoRI fragment of v-Ki-ras, 2.9 kb SacI fragmentof the human c-Ha-ras1 gene, and a BamHI 4.5 kb fragment of the c-mycgene. Glyceraldehyde phosphate dehydrogenase cDNA [Dugaiczyk, A., J. J.Haron, E. M. Ston, O. E. Dennison, K. N. Rothblum, and R. J. Schwartz.1983. Cloning and sequencing of a deoxyribonucleic acid copy ofglyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acidisolated from chicken muscle. Biochem. 22:1605-1613] was provided by M.A. Tainsky (University of Texas, Houston), and a mouse transin cDNA byG. T. Bowden (University of Arizona, Tucson). The cDNA probe for mousehistocompatibility class I antigens was a gift from G. Jay (NIH,Bethesda). Radiolabeled probes were prepared with [³² P]dCTP (NEN) usinga random primed DNA labeling kit (Boehringer Mannhelm, Germany).

In Vitro Carcinogenesis Induced by 5AzadC and Its Prevention byPhenylacetate.

Untreated 4C8 and PR4 formed contact-inhibited monolayers composed ofepithelial-like cells. In agreement with previous observations,transient exposure of these cultures to 0.1 uM 5AzadC during logarithmicphase of growth resulted in rapid and massive neoplastic transformation.Within one week of 5AzadC treatment, the great majority of the cellpopulation became refractile and spindly in shape, and formedmultilayered cultures with increased saturation densities (Table 7),indicative of loss of contact inhibition of growth. These phenotypicchanges could be prevented by the addition of 5-10 mM NaPA (Table 7).Several different regimens of NaPA treatment were found to be similarlyeffective. These included: (a) pre-treatment with NaPA, starting one dayprior to the addition of 5AzadC; (b) simultaneous exposure to bothdrugs, and; (c) addition of NaPA one day after 5AzadC. In all cases,cells were subsequently subjected to continuous treatment with NaPA forat least one week. Cells cultured under these conditions, like thosetreated with NaPA alone, formed contact-inhibited monolayers resemblinguntreated controls. These cells maintained the benign growth pattern forat least three weeks after NaPA treatment was discontinued.

That NaPA prevents neoplastic transformation was further indicated bythe inability of cells to invade reconstituted basement membranes(matrigel), and form tumors in athymic mice. When plated onto matrigel,5AzadC-transformed 4C8 and PR4 cells developed net-like structurescharacteristic of highly malignant cells, and eventually degraded theextracellular matrix components. In marked contrast, NaPA-treatedcultures formed small, non-invasive colonies on top of the matrigel, aspreviously observed with normal fibroblasts. Untreated parental cellsexhibited an intermediate phenotype, as their colonies were slow growingand non-invasive, yet irregular in shape possibly due to increased cellmotility. The chemopreventive effect of phenylacetate could be mimickedby its precursor, phenylbutyrate. Cells exposed to 5AzadC in thepresence of sodium phenylbutyrate (NaPB, 1.5-3 mM) maintained contactinhibited growth and exhibited a benign phenotype when placed ontomatrigel (Table 7).

                  TABLE 7                                                         ______________________________________                                        Effect of 5AzadC and                                                          NaPA on DNA Methylation                                                                       DNA Methylation                                               Cells   Treatment.sup.a                                                                             % 5mC.sup.b                                                                             % of Control                                  ______________________________________                                        4C8     none          3.49 ± 0.06                                                                          100                                                   5AzadC        1.52 ± 0.27                                                                          43                                                    NaPA          2.22 ± 0.10                                                                          63                                                    5AzadC + NaPA 1.62 ± 0.18                                                                          46                                            PR4     none          2.72 ± 0.16                                                                          100                                                   5AzadC        1.11 ± 0.22                                                                          41                                                    NaPA          1.25 ± 0.08                                                                          46                                                    5AzadC + NaPA 1.06 ± 0.11                                                                          39                                            ______________________________________                                         .sup.a Cells were treated with 0.1 uM 5AzadC and/or 10 mM NaPA and the        percentage of 5 mC was determined as described in "Materials and Methods"     .sup.b Data indicate the mean ± S.D. (n = 4) of two experiments.      

The in vitro growth characteristics of cells correlated with theirbehavior in athymic mice. 5AzadC-treated 4C8 cells developed rapidlygrowing fibrosarcomas within 2 weeks of s.c. transplantation into mice.Consistent with their behavior in vitro, the parental cells were farless aggressive, forming small lesions after 3-4 weeks in three of eightrecipient animals. However, no tumors developed in animals injected with4C8 cells that had been pre-treated for one week in culture with thecombination of 5AzadC and NaPA (Table 7). There was also no tumorformation in mice injected with 4C8 treated with NaPA alone. Thereforeit follows that NaPA induced phenotypic reversion of the premalignantfibroblasts and prevented their malignant conversion the cytosineanalog.

Modulation of Gene Expression by NaPA.

The NIH 3T3-derived cells lines, 4C8 and PR4, carry an LTR-activatedc-Ha-ras protooncogene. Northern blot analysis of 5AzadC-treated 4C8revealed a significant increase in ras mRNA levels and a decline in thedifferentiation marker, collagen α (type I) transcripts. No such changesin gene expression occurred in cultures to which NaPA was added.Withdrawal of NaPA after one week of continuous treatment did not causerestoration of ras expression, confirming that the therapeutic benefitof NaPA is stable in the absence of further treatment.

Effect of Phenylacetate and 5AzadC on DNA methylation.

5AzadC is a potent inhibitor of DNA methylation, an epigenetic mechanismimplicated in the control of gene expression and cell phenotype.Hypomethylation may underlay the therapeutic effect of 5AzadC in cancerand in severe inborn anemias [Momparler, R. L., G. E. Rivard, and M.Gyger. 1985. Clinical trial on 5-aza-2'-deoxycytidine in patients withacute leukemia. Pharmac. Ther., 30:277-286; Stamatoyannopoulos, J. A.,and A. W. Nienhuis. 1992. Therapeutic approaches to hemoglobin switchingin treatment of hemoglobinopathies. Annu. Rev. Med., 43:497-521; Ley, T.J., J. DeSimone, N. P. Anagnou, G. H. Keller, R. K. Humphries, P. H.Turner, P. H., N. S. Young, P. Heller, and A. W. Nienhuis. 1982.5-Azacytidine selectively increases gamma-globin synthesis in a patientwith beta⁺ thalassemia. N. Engl. J. Med. 307:1469-1475]. However,changes in DNA methylation could also be responsible for itscarcinogenic potential. It was of interest therefore to determine thedegree of DNA methylation in cells protected by phenylacetate. As wouldbe expected, 5AzadC caused a significant decrease in the content of5-methylcytosine (5mC) (Table 6). There was, however, a comparabledecline in 5mC in cells treated with 5AzadC in combination with NaPA, aswell as in those treated with NaPA alone (Table 6).

                                      TABLE 6                                     __________________________________________________________________________    In vitro Prevention by                                                        Phenylacetate of 5AzadC-Induced Carcinogenesis                                Cell     Saturation Density.sup.a                                                                Invas-                                                                             Tumorigenicity in mice.sup.c                          Treatment                                                                              (cells/cm.sup.2 × 10.sup.-5)                                                      iveness.sup.b                                                                      Incidence                                                                          Tumor Size (mm)                                  __________________________________________________________________________    None     3.9       -    3/8   1.0 (0.5-2)                                     5AzadC   7.0       +    8/8  11.5 (4-19)                                      5AzadC + NaPA                                                                          1.6       -    0/8  0                                                5AzadC + NaPB                                                                          1.1       -    ND                                                    NaPA     ND        -    0/8  0                                                NaPB     1.3       -    ND                                                    __________________________________________________________________________     .sup.a Cell were treated simultaneously with the indicated drugs and kept     in culture for 5 days post confluency at which time they were detached an     counted. Exposure to 5AzadC was transient as described in Materials and       Methods, while treatment with NaPA and NaPB continued throughout the          experiment. Similar results were obtained when NaPA treatment was             initiated one day prior or after cell exposure to 5AzadC (data not shown)     .sup.b Cells were plated on top of a matrigel layer and observed for          malignant growth pattern, i.e., development of characteristic processes       and degradation of the reconstituted basement membrane and invasion           towards the plastic surface below.                                            .sup.c Cells pretreated in culture were injected s.c (5 × 10.sup.5      cells per site) into 2 month old female athymic nude mice. Results            determined after 3 weeks indicate tumor incidence (tumor bearing, injecte     animals) and size. The values of tumor size are mean (range).                 ND = not determined.                                                     

In Vivo Chemoprevention by NaPA.

To determine the efficacy of NaPA in vivo, studies were extended toinclude an animal model involving athymic mice bearing thenon-tumorigenic 4C8 cells transplanted subcutaneously. A single i.p.injection of mice with 5AzadC (20 mg/kg) resulted in tumor developmentat the site of 4C8 cell inoculation. However, when mice were pre-treatedwith NaPA 1.5 hr prior to 5AzadC injection, and NaPA treatment continuedfor 22 days thereafter, the incidence of tumor formation wassignificantly decreased (Table 8). There were no adverse effectsassociated with NaPA treatment as indicated by animal weight andbehavior. Further more, despite causing DNA hypomethylation NaPA did notinduce neoplastic transformation of transplanted 4C8 cells. Animalsprotected by NaPA either failed to develop tumors or formed slow-growinglesions at the site of 4C8 inoculation. The animal data is consistentwith the in vitro findings, indicating that NaPA can prevent5AzadC-induced neoplastic transformation without producing significanttoxicities.

                  TABLE 8                                                         ______________________________________                                        In vivo Chemoprevention                                                       by Phenylacetate                                                                    Animal        Tumor Incidence.sup.b                                                                      Tumor Size.sup.c                             Group Treatment.sup.a                                                                             positive/total                                                                             mean (range)                                 ______________________________________                                        A     PBS           0/4          0                                            B     NaPA          0/4          0                                            C     5AzadC + PBS  9/9          12 (2-29)                                    D     5AzadC + NaPA 4/10         3 (0-10)                                     ______________________________________                                         .sup.a 4C8 cells (5 × 10.sup.5 per site) were transplanted s.c. int     athymic mice. The next day, the animals in were treated i.p. with 400         mg/kg NaPA, and 1.5 hr later with 20 mg/kg 5AzadC. NaPA treatment was         repeated at 4.5 hours following 5AzadC injection. Subsequent treatments       involved NaPA injections twice daily for 8 days, and once a day for           additional 2 weeks. PBS was used as a control.                                .sup.b Data indicates tumor growth at 4 weeks after 5AzadC treatment.         Spontaneous tumors developed thereafter in control animals receiving PBS,     and subsequently in those treated with NaPA.                                  .sup.c Tumor diameter in millimeters.                                    

There is considerable interest in the use of non-toxic differentiationinducers in cancer chemoprevention. Drug toxicity is particularlyimportant considering the overall health condition and variablelife-span of candidate populations, i.e., high-risk individuals andpatients in remission. The differentiation inducer phenylacetate canprevent 5AzadC-induced carcinogenesis both in vitro and in vivo whenused at nontoxic doses.

Chemoprevention can be accomplished by either blocking the "initiation"step of carcinogenesis (i.e., mutagenesis), or by suppressing"promotion" and progression to malignancy. The current studies, usingpremalignant cells with an activated ras oncogene as a model, examinedthe efficacy of phenylacetate as an anti-promotional drug. Other wellcharacterized chemopreventive agents that block promotion includevitamin A and its synthetic retinoids; like phenylacetate, thesecompounds are also regulators of cell growth and differentiation.

The current studies exploited in vitro and in vivo models involvingfibroblasts (designated 4C8 and PR4) that are highly vulnerable tomalignant conversion by the DNA hypomethylating agents 5AzadC and 5AzaC(16, 17). Transient exposure of these cells to 5AzadC, either in cultureor in recipient athymic mice, caused rapid neoplastic transformation.Malignant conversion was associated with an increase in ras mRNA levelsand down-regulation of collagen type I expression, indicating loss ofcell differentiation. These profound biological and molecular changesbrought about by 5AzadC are prevented by a simultaneous treatment withnon-cytotoxic concentrations of phenylacetate and its precursor,phenylbutyrate. Phenylacetate's antitumor activity and lack of toxicitywere confirmed in athymic mice. In the in vivo model, mice bearing thesusceptible 4C8 cells transplanted s.c. were injected i.p. with 5AzadC.All mice so treated developed rapidly growing fibrosarcomas; however,the incidence of tumor formation was markedly reduced by systemictreatment with NaPA.

The mechanism by which NaPA prevented the 5AzadC induced malignantconversion is unclear. Like other chemopreventive agents that blockpromotion, phenylacetate may act by inducing cytostasis and tumormaturation. There is a growing body of evidence indicating thatphenylacetate can cause selective growth arrest and tumordifferentiation in vitro and in rodent models. In some cases, e.g.,promyelocytic leukemia, differentiation induced by phenylacetate waslinked to a decline in myc oncogene expression. In NaPA-treated 4C8,protection from de-differentiation (evidenced by growth characteristicsand collagen expression), was associated with inhibition of rasoverexpression. Down-regulation of oncogene expression may thus beresponsible in part for the chemopreventive activity of NaPA. Inaddition to affecting ras at the mRNA levels, phenylacetate, aninhibitor of the mevalonate pathway of cholesterol synthesis [Castillo,M., J. Iglesias, M. F. Zafra, and E. Garcia-Peregrin. 1991. Inhibitionof chick brain cholesterolgenic enzymes by phenyl and phenolicderivatives of phenylalanine. Neurochem. Int., 18: 171-174], could alsoblock the post-translational modification of the ras-encoded protein,p21. Limonene, an inhibitor of p21 prenylation, is a chemopreventiveagent as well.

Phenylacetate blocked carcinogenesis by 5AzadC despite the decline in5mC content. In fact, NaPA itself was found to inhibit DNA methylation;yet, in contrast to 5AzadC, NaPA was not carcinogenic. Correlationsbetween carcinogenic potential and DNA hypomethylating activities ofchemical agents have been previously documented in tissue culturemodels, and alterations in DNA 5mC patterns were proposed to contributeand enhance the initiation of carcinogenesis. However, the present dataindicate that quantitative changes in DNA methylation alone are notsufficient to affect cell phenotype and thus, hypomethylating activityis not sufficient to induce the tumorigenic phenotype in these in vitroand animal models.

The selective induction of specific genes by intracellular factors andchemical agents subsequent to demethylation has been reported by severallaboratories. For example, an increase in human gamma-globin geneexpression in vitro was found to require activation byhexamethylenebisacetamide following treatment with 5AzaC [Ley J. T., Y.L. Chiang, D. Haidaris, N. P. Anagnou V. L. Wilson, and W. F. Anderson.1984. DNA methylation and regulation of the human β-globin like genes inmouse erythroleukemia cells containing human chromosome 11. Proc. Natl.Acad. Sci. USA. 81:6618-6622]; demethylation of the gene by 5AzaC wasnot sufficient for gene expression. By contrast, phenylacetate andphenylbutyrate induced gamma-globin gene expression with subsequentaccumulation of fetal hemoglobin in cultured erythroid progenitors andin humans. In addition to affecting DNA methylation, NaPA and NaPB alsoactivate a nuclear receptor that functions as a transcriptional factor(the peroxisome proliferator receptor is discussed herein). Thus, onepossible explanation for the differences in carcinogenic opposingactivities between NaPA/NaPB and 5AzadC seen here may be the ability ofthe aromatic fatty acids to induce the expression of genes critical togrowth control. Phenylacetate and related compounds can possibly reversethe methylation-mediated state of repression of silent anti-oncogenes.The finding of DNA hypomethylation by NaPA in mammalian cells does notcome as a surprise in view of previous studies demonstrating that, atmillimolar concentrations, phenylacetate inhibits DNA methylation inplant. Interestingly, at such high concentrations, phenylacetate alsoinhibits plant tumor cell proliferation. Therefore, the effect ofphenylacetate on DNA methylation and its role in regulating growth anddifferentiation have been conserved in evolution.

The outcome of combining NaPA with 5AzadC (or 5AzaC) is of particularinterest. The cytosine analogs have been shown to benefit patients withsevere blood disorders such as leukemia, sickle cell anemia, andβ-thalassemia. There is now experimental data suggesting that 5AzadC maybe active also in some solid tumors, including malignant melanoma (Weberet al, submitted) and prostate carcinoma. Unfortunately, the clinicalapplication of 5AzadC has been limited by concerns regardingcarcinogenesis. The data indicate that NaPA can minimize thecarcinogenic risk, while both preserving and potentiating thetherapeutic effects of 5AzadC. Studies with human leukemic cells andwith erythroid progenitors derived from patients withβ-hemoglobinopathies revealed that NaPA can enhance the efficacy of5AzadC, causing superinduction fetal hemoglobin production. Moreover,the addition of NaPA/NaPB to nontoxic, yet sub-optimal concentrations of5AzadC, induced complete growth arrest and promoted apoptosis incultured hormone-refractory prostatic carcinoma cells (unpublisheddata).

It appears therefore that phenylacetate, a common amino acid derivative,may be of value as an antitumor and chemopreventive agent. NaPA, whichhas an unpleasant odor, can be substituted by its precursor, NaPB (or aderivative or analog of NaPB), for oral administration. Upon ingestionby humans, phenylbutyrate undergoes β-oxidation to phenylacetate. LikeNaPA, NaPB exhibits antitumor and chemopreventive activities inexperimental models, and both drugs already proved safe for long-termoral treatment of children with urea cycle disorders. More recentclinical studies involving adults with cancer have confirmed thatmillimolar plasma levels of phenylacetate and phenylbutyrate can beachieved with no significant adverse effects. NaPB/NaPA will benefithigh risk individuals predisposed to cancer development, be applied incombination with other anticancer therapeutics to enhance efficacy andminimize adverse effects, and perhaps be used in maintenance therapy toprevent disease relapse.

EXAMPLE 10

HbF induction in K562 cells by NaPA and derivatives

The K562 erythroleukemia line serves as a model for inherited anemiasthat are associated with a genetic defect in the beta globin geneleading to severe β-chain hemoglobinopathies.

The results reported in Table 9 also show that there is a synergisticaffect when leukemia cells are exposed NaPA in combination withinterferon alpha, a known biological response modifier or with thechemotherapeutic drug hydroxyurea (HU).

                  TABLE 9                                                         ______________________________________                                        Induction of Hemoglobulin Synthesis                                           in Erythroleukemia K562 cells*                                                                   POSITIVE  CELL                                                                CELLS     VIABILITY                                        TREATMENT BENZIDINE                                                                              (%)       (%)                                              ______________________________________                                        Control            1.8       >95                                              NaPA                                                                          0.8 mg/ml          6.0                                                        1.6 mg/ml          17.1                                                       Interferon 500     13.5                                                       IU/ml                                                                         HU 100 uM          17.2                                                       NaPA (0.8 mg/ml)   40-42                                                      + HU or IFN                                                                   ______________________________________                                         *Results at seven days of treatment.                                     

Analysis of gene transcripts showed accumulation of mRNA coding forgamma globin, the fetal form of globin. This was confirmed at theprotein level.

Using the erythroleukemia K562 cell line described above it was foundthat 4-hydroxyphenylacetate was as effective as NaPA in inducing fetalhemoglobin accumulation, but was less inhibitory to cell proliferation.In contrast, some other analogs such as 2,4- or3,5-dihydroxyphenylacetate were found to be highly toxic.

EXAMPLE 11

PC3 and DU145 cells--NaPA as an antitumor agent

The effectiveness of NaPA as an antitumor agent was further evaluated ina variety of experimental models. Studies in depth were performed withtwo androgen-independent human prostate adenocarcinoma cell lines, PC3and DU145, established from bone and brain metastases, respectively, aswell as hormone responsive LNCaP cultures. NaPA treatment of theprostatic cells resulted in concentration-dependent growth arrest,accompanied by cellular swelling and accumulation of lipid that stainedpositive with Oil-Red O. The results of this study are shown in FIG. 11.As illustrated therein, an IC₅₀ for NaPA occurred at 600-800 μg/ml.Significantly higher doses were needed to affect the growth of activelyreplicating normal human FS4 skin fibroblasts or normal endothelialcells (IC₅₀ from 12-15 μM), indicating a selective cytostatic effect ofthe drug.

EXAMPLE 12

PC3 cells--non-invasiveness after NaPA treatment

It is known that PC3 cells are invasive in vitro and metastatic inrecipient athymic mice. [Albini, A. et al. A rapid in vitro assay forquantitating the invasive potential of tumor cells. Cancer Res.47:3239-3245 (1987)]. The invasiveness of PC3 cells which is indicativeof their malignant phenotype can be assessed by their ability to degradeand cross tissue barriers such as matrigel, a reconstituted basementmembrane. Untreated PC3 cells and PC3 cells treated with NaPA for 4 daysin culture were quantitatively analyzed in a modified Boyden chambercontaining a matrigel-coated filter with FS4 conditioned medium as achemoattractant. After 4 days of treatment with 800 μg/ml of NaPA inT.C. plastic dishes, 5×10⁴ cells were replated onto 16 mm dishes(Costar, Cambdrige, Mass.) coated with 250 μl of matrigel 10 mg/ml.Control showed the characteristic growth pattern of untreated cells,i.e, formation of net-like structures composed of actively replicatingcells which eventually degraded the matrigel and formed monolayers onthe plastic surface beneath. In contrast to the controls, the NaPAtreated cells formed isolated small colonies which resembled normalhuman FS4 cells 8 days after plating. The NaPA treated cells failed todegrade the matrigel barrier. The formation of small noninvasivecolonies on top of the matrigel is indicative of loss of malignantproperties following treatment. Results of the in vitro invasion assayscorrelate highly with the biological behavior of cells in vivo.

EXAMPLE 13

PC3 cells--PAG treatment did not hinder invasiveness

PC3 cells treated with NaPA for one week in culture, in contrast tountreated cells or those treated with PAG, failed to form tumors whentransplanted s.c. into athymic mice. These results are shown in Table10.

                  TABLE 10                                                        ______________________________________                                        Tumorigenicity of Prostatic PC3                                               Cells in Nude Mice                                                            TREATMENT            Diameter   Weight                                        (mg/ml)   Incidence  (mm ± S.D.)                                                                           (mg ± S.D.)                                ______________________________________                                        None      7/7        9 ± 3   285 ± 60                                   NaPA 0.8  1/7        2          50                                            PAG 0.8   3/4        8 ± 2   245 ± 35                                   ______________________________________                                    

PC3 cells were pretreated for 1 week in culture and then injected (2×10⁵cells/animal) s.c. into 4-5 week-old female athymic nude mice. Theresults in Table 10 indicate the incidence of tumor bearinganimals/injected animals as well as tumor size measured as meandiameter±S.D. 8 weeks later.

EXAMPLE 14

Phenylacetate in combination with suramin

To further substantiate the phenotypic changes observed in the NaPAtreated prostatic PC3 cells, Northern blot analysis revealed that NaPAinhibited the expression of collagenase type IV, one of the majormetalloproteases implicated in degradation of basement membranecomponents, tumor cell invasion, and metastasis. Furthermore, it wasfound that NaPA treated prostatic PC3 cells showed an increase in thelevel of HLA-A mRNA which codes for major histocompatibility class Iantigen known to affect tumor immunogenicity in vivo.

The malignant prostatic cell lines exhibit numerous abnormalities ingene expression, including increased production of autocrine tumorgrowth factor-β (TGF-β) and elevated activity of urokinase plasminogenactivator (uPA). Members of the TGF-β family have been implicated intumor growth control, anglogenesis, and immunosuppression. uPA, incontrast, is a serine protease involved in degradation of extracellularstroma and basal lamina structures, with the potential to facilitatetumor invasion and metastasis. It was of interest, therefore, to examinethe effect of NaPA on TGF-β and uPa expression in the prostatic tumorcells. Northern blot analysis of PC3 after 72 h treatment revealed adecrease in TGF-β2 mRNA levels; the effect was specific for TGF-β2 asthere was no change in the expression of TGF-β1. The decrease in TGF-β2was accompanied by approximately a twofold increase in the levels ofHLA-A3 mRNA, as previously observed in treated human leukemic HL-60cells.

Preliminary analysis of uPA transcript levels showed no significantchange after NaPA treatment. There was, however, a reductioncell-surface uPA activity. The hormone-refractory malignant PC3 andDU145 cells, but not the more indolent hormone-responsive LNCaP,displayed high cell-bound uPA activity. Because the parental PC3cultures are composed of highly heterogenous cell populations withrespect to uPA production, more homogeneous subclones were establishedby limiting dilutions and single-cell cloning. A subclone designatedPC3-1, which resembled the parental PC3 cells in its invasive capacityand surface-localized uPA activity (2.2±0.3×10⁶ Plau units per cell),was chosen for further studies. After 3 d of treatment of PC3-1 withNaPA 5 mM there was over 50% reduction in cell-associated uPA activity;the effect was dose-dependent and reversible upon cessation oftreatment. Similar results were obtained with DU145 cells. Assayspecificity was confirmed by the fact that pretreatment of cells withneutralizing anti-human uPA monoclonal antibodies, or addition ofantibodies at the time of assay, blocked over 95% of theplasminogen-dependent proteolytic activity. Plasminogen-independentproteolysis constituted 30% of the maximal fibronectin degradingactivity, and was similar for both NaPA-treated cells and untreatedcontrols.

NaPA in Combination with Suramin

                  TABLE 11                                                        ______________________________________                                        Malignant Melanoma A375                                                       Treatment           Growth      Viability                                     (μg/ml)          (% of control)                                                                            (%)                                           ______________________________________                                        None                100         >95                                           NaPA 400            63.3        >95                                           Suramin                                                                       38                  78.3        >95                                           75                  56.8        >95                                           150                 38.6         92                                           300                 26.6         82                                           NaPA (400) + Suramin (38)                                                                             45.5        >95                                                  + Suramin (75)                                                                             30.1         94                                                  + Suramin (150)                                                                            21.8         92                                       ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Prostate Adenocarcinoma PC3                                                   Treatment           Growth      Viability                                     (μg/ml)          (% of control)                                                                            (%)                                           ______________________________________                                        None                100         >95                                           NaPA 800            59.6        >95                                           Suramin                                                                       75                  58.5        nd                                            150                 46.5        nd                                            300                 31.0        nd                                            NaPA (800) + Suramin (75)                                                                             24.2        90                                                   + Suramin (150)                                                                            10.9        64                                        ______________________________________                                    

NaPA was found to significantly potentlate the therapeutic effect ofsuramin, the only experimental drug known to be active against prostatecancer.

However, drug toxicities have been a major concern. In agreement withprevious in vitro studies, we found that toxic doses of suramin (300μg/ml) were needed in order to achieve over 50% inhibition of prostaticDU145 cell growth. This cellular model was used to examine whether NaPAcould enhance the activity of suboptimal but less toxic doses ofsuramin. Results of this examination show that NaPA and suramin act inan additive manner to inhibit DU145 cell proliferation. Moreover,suramin was found to be significantly more active if added toglutamine-depleted medium. Despite significant differences in tumorsensitivities, there was complete growth arrest when DU145 and PC3 cellswere treated for 6 d with both NaPA and suramin in glutamine-depletedmedium, under conditions in which each treatment alone had only apartial effect. Similarly, Tables 11 and 12 show the effect of combinedNaPA and suramin treatment of malignant melanoma A375 cells and prostateadenocarcinoma PC3 cells.

It is known that a disease state characterized by the presence of benignhyperplastic lesions of the prostate exists as a separate disease entityand has been identified in many patients that progress to a diagnosis ofprostatic cancer. Based on the above, it is anticipated that NaPA, inaddition to being effective in the treatment of prostatic cancer, wouldbe effective in treating patients having benign hyperplastic prostaticlesions.

Further experiments demonstrated that NaPA appears to have broadantitumor activity affecting a wide spectrum of malignancies. Theexperimental data presented in Table 13 indicate that NaPA 0.4-0.8 mg/mlcaused about 50% inhibition of growth in treated adenocarcinoma of theprostate cell lines PC3 and DU145, melanoma A375 and SK MEL 28, lungadenocarcinoma H596 and H661, and astrocytoma U87, U373, and 343.Somewhat higher concentrations (1.0-1.5 mg/ml) were needed to cause asimilar inhibition of squamous cell carcinoma A431, breast tumor MCS-7,osteosarcoma KRIB, and fibrosarcoma V7T. Typically, NaPA treatment wasassociated with growth arrest, induction of differentiation markers,reduced invasiveness in vitro and loss of tumorigenicity in nude mice.

                  TABLE 13                                                        ______________________________________                                        Responses of Different Tumor Ceil Lines                                       to NaPA Treatment                                                                                   % Inhibition by                                         #        Tumor Cell Line                                                                            NaPA 0.8 mg/ml.sup.a                                    ______________________________________                                        1        Melanoma                                                                      A375         ≧70                                                       SK MEL 28    >50                                                     2        Prostatic Ca.sup.b                                                            PC3          ≧50                                                       DU145        ≧50                                                       LaNCop       >50                                                     3        Astrocytoma                                                                   U87          ≧50                                                       U343         ≧50                                                       U373         ≧50                                              4        Kaposi's Sarcoma                                                              KS           ≦40                                              5        Leukemia HL-60                                                                             ≦40                                              6        Leukemia K562                                                                              ≦30                                              7        Breast Cancer                                                                 MCF-7        ≦30                                              8        Osteosarcoma                                                                  KRIB         ≦30                                                       HOS          <20                                                     9        Fibrosarcoma                                                                  V7T          ≦30                                                       RS485        ≦30                                               10      Squamous Cancer                                                               of Head and Neck                                                              A431         <30                                                     ______________________________________                                         .sup.a Pharmacologically attainable concentration                             .sup.b Carcinoma                                                         

Of major interest in Table 13 are the following:

#1-3 Tumor cells show significant response i.e., ≧50% inhibition ofproliferation within one week of treatment. Cf. FIG. 15.

#4 KS, an HIV-associated disorder, may be more dramatically affected byNaPA in humans, due to inhibition of HIV expression and of essentialgrowth factors released by infected lymphocytes.

#5,6 The treated HL-60 promeyelocytic leukemic cells undergo terminaldifferentiation, a desirable outcome of chemotherapy. In the K562erythroleukemia, NaPA induced reversible erythroid differentiation withno significant growth arrest (<30%); thus the K562 data is of interestwith respect to treatment of certain anemias, not cancer.

Less attractive:

#7-10 For effective responses, the tumors may require much higher drugconcentrations if used alone.

Although some of the malignant cell lines seem more sensitive thanothers, all were significantly more affected by NaPA when compared tonormal or benign cells. For example, NaPA inhibited the growth ofmalignant osteosarcoma (KRIB) cells more so than benignosteosarcoma-derived HOS cells. A differential effect was seen also inras-transformed fribrosarcoma V7T, when compared to the parentalnon-tumorigenic NIH 3T3 cells. As to normal human cells, as much as 2-4mg/ml of NaPA were needed to cause a significant inhibition of growth toprimary human skin FS4 fibroblasts. It should be noted that thetreatment was not toxic to either the malignant or the normal cells.

The concentration range found to selectively suppress malignant growthcan be readily obtained in the clinical setting without causingsignificant side effects. Intravenous infusion of NaPA into humans at250-500 mg/kg/day which results in plasma levels of 600-800 μg/ml hasbeen found to be a well tolerated treatment. Cytotoxicity in tissueculture was observed when the NaPA concentration was as high as 3 mg/mlor higher.

EXAMPLE 15

Phase I clinical trials

Patient Population.

Patients were eligible for this study if they had advanced solid tumorsfor which conventional therapy had been ineffective, a Karnofskyperformance status greater than 60%, normal hepatic transaminases andtotal bilirubin, a serum creatinine less than 1.5 mg/dl, and normalleukocyte and platelet counts. All patients signed an informed consentdocument that had been approved by the National Cancer Institute (NCI)Clinical Research Subpanel. Seventeen patients, 16 men and 1 woman, witha median age of 57 years (range: 36-75) were enrolled between Januaryand June 1993. Disease distribution included progressive, metastatic,hormone-refractory prostate cancer (9 patients), anaplastic astrocytomaor glioblastoma multiform (6 patients), ganglioglioma (1 patient) andmalignant pleural mesothelioma (1 patient).

Drug Preparation and Administration.

Sodium phenylacetate for injection was prepared from bulk sodiumphenylacetate powder supplied by Elan Pharmaceutical Research Co.(Gainesville, Ga.). The finished injectable stock solution wasmanufactured by the Pharmaceutical Development Service, PharmacyDepartment, Clinical Center, NIH, in vials containing sodiumphenylacetate at a concentration of 500 mg/ml in sterile water forinjection, USP, with sodium hydroxide and/or hydrochloric acid added toadjust the pH to approximately 8.5. Doses of sodium phenylacetate to beinfused over 30 minutes to 2 hours were prepared in 150 ml of sterilewater for injection, USP. Doses of phenylacetate to be given over 24hours were prepared similarly to yield a total volume of 1,000 ml andwere administered using an infusion pump.

The protocol as originally designed delivered an i.v. bolus dose ofphenylacetate (150 mg/kg over 2 hours) on the first day of therapy, toallow for the estimation of pharmacokinetic parameters. This wasfollowed 24 hours later by a CIVI of the drug for the next 14 days.Cycles of two week drug infusions were repeated every 6 weeks. The rateof drug infusion was to be increased in sequential cohorts of at leastthree patients, and individual patients could escalate from one doselevel to the next with sequential cycles of therapy provided they hadexperienced no drug-related toxicity and their disease was stable orimproved.

The protocol underwent several modifications over the 6 month period.First, the size of the initial bolus dose was reduced from 150 to 60mg/kg i.v. and the bolus infusion duration from 2 hours to 30 minutes,after the first three patients were treated. This change resulted indrug concentrations optimal for estimating the drug's pharmacokinetics(vide infra) within a six hour time period. Second, after the non-linearnature of phenylacetate's pharmacokinetics was recognized (vide infra),the protocol was changed from a fixed dose escalation (dose levels 1 and2:150 and 250 mg/kg/day, respectively) to a concentration-guidedescalation trial (dose levels 3 and 4:200 and 400 μg/ml, respectively).In the latter format each patient was given an i.v. bolus dose ofphenylacetate (60 mg/kg over 30 minutes) one week prior to beginning a14 day CIVI of the drug. The patient-specific pharmacokinetic parametersestimated from the bolus dose were used to calculate an infusion ratethat would maintain the serum phenylacetate concentration at thetargeted level during the 14 day infusion. Serum drug concentrationswere measured weekly, prompting weekining reestimation of individualpharmacokinetics and dosage adjustment (adaptive control with feedback).

Sampling Schedule.

With the initial 150 mg/kg i.v. bolus, blood samples were obtainedthrough a central venous catheter at the following timepoints calculatedfrom the beginning of the infusion: 0, 60, 115, 125, 135, 150, 165, 180,240, 360, 480, and 600 minutes. For the 60 mg/kg bolus given over 30minutes, blood sampling was performed at 0, 30, 60, 75, 90, 105, 120,150, 180, 270 and 390 minutes from the beginning of the infusion. Atdose levels 1 and 2, blood samples were obtained daily during the CIVI,while at dose levels 3 and 4, blood samples were obtained on days 1, 2,3, 8, 9 and 10 of the infusion. Twenty-four hour urine collections forthe determination of phenylacetate and phenylacetylglutamine excretionwere obtained on days 1, 7 and 14 of therapy. Sampling of the CSF wasperformed only if clinically indicated.

Determination of sodium phenylacetate and phenylacetylglutamine in serumand urine by high performance liquid chromatography (HPLC).

Blood was drawn by venipuncture into a Vacutainer® tube free ofanticoagulant and was then refrigerated. It was centrifuged at 1,200 gfor 10 minutes in a Sorvall® RT 6000D centrifuge (DuPont Co.,Wilmington, Del.) at 4° C. Serum was then removed and stored in NuncCryotubes (Nunc Co., Denmark) at -70° C. until the day of analysis.

A standard curve was generated by adding known amounts of sodiumphenylacetate (Elan Pharmaceutical Research Co., Gainesville, Ga.) andphenylacetylglutamine (a gift from Dr. S. W. Brusilow, Johns HopkinsUniversity, Baltimore) to a commercial preparation of pooled serum(Baxter Healthcare Corporation, Deerfield, Ill.). The standard valuesspanned the expected range of serum concentrations: 0, 5, 10, 20, 50,100, 250, 500, 750 and 1,500 μg/ml.

Two hundred microliters of serum were pipetted into a 1.7 ml Eppendorfttube (PGC Scientifics, Gaithersburg, Md.). Protein extraction wascarried out by adding 100 μl of a 10% (v/v) solution of perchloric acid(Aldrich Chemical Co., Milwaukee, Wis.). The tube was vortexed and thencentrifuged at 4,500 g for 10 minutes. One hundred and fifty microlitersof supernatant were transferred to a new 1.7 ml Eppendorf tube and 25 μlof 20% KHCO₃ (w/v) was added to neutralize the solution. This wascentrifuged at 4,500 g for 10 minutes and 125 μl of supernatant weretransferred to an autosampler vial and maintained at 10° C. until HPLCinjection. Urine samples were processed in an identical manner after aninitial 1:10 dilution with water.

The HPLC system (Gilson Medical Electronics, Middleton, Wis.) wascomposed of two pumps (305 and 306), an 805 manometric module, an 811Cdynamic mixer, a 117 variable wavelength UV detector and a 231autosampler fitted with a 20 μl injection loop and cooled with a GreyLine model 1200 cooling device. The column was a Waters® (MilliporeCorporation, Milford, Mass.) C18 Nova-Pak, 3.9×300 mm, maintained at 60°C. with a Waters® temperature control module. The mobile phase solutionsconsisted of fifty microliter samples were auto-injected onto a 10 cmcation-ion exchange column integrated into a Beckman Model 6300 AminoAcid Analyzer (Beckman Instruments Inc., Palo Alto, Calif.). The solventflow rate (2:1 water/ninhydrin) was maintained constant at 0.5 ml/min.Column temperature was raised by 1.5° C. per minute to elute sarcosine,the internal standard. The column was regenerated with lithium hydroxideat 70° C. following each injection. Absorbance was measured at 570 nmand 440 nm following post-column color development with ninhydrin-RX(Beckman Instruments Inc., Palo Alto, Calif.) at 131° C. Beckman SystemGold software was used for data acquisition and data management.

Pharmacokinetic Methods.

Initial estimates of V_(max) and K_(m) for phenylacetate were obtainedby generating Lineweaver-Burk plots from concentration versus timecurves following i.v. bolus doses. These initial parameter estimateswere refined by non-linear least squares fitting, using the Nelder-Meaditerative algorithm, as implemented in the Abbottbase® PharmacokineticSystems software package (Abbott Laboratories, Abbott Park, Ill.,version 1.0).

Statistical Methods.

The Student's t-test was used to compare estimates of phenylacette'spharmacokinetic parameters derived from the Lineweaver-Burk plots withthose obtained using non-linear given set of dosing and concentrationdata was quantified by calculating the weighted sum of the errorssquared following non-linear least-squares fitting. The standarddeviation of the errors was modeled as a function of drug concentrationmultiplied by the coefficient of variation of the assay. Confidenceregions for the parameters were derived from the weighted sum of squaresin the model incorporating the induction parameters, and approximatesignificance levels for testing between the two models were calculatedusing the F distribution [Draper, N. R., Smith H. Applied RegressionAnalysis. New York, John Wiley and Sons, p. 282, 1966]. The significancelevels of individual cycles were analyzed by the Spearman rankcorrelation method in an attempt to discern whether a relationshipexisted between time-dependent changes in drug clearance and dose.

Analytical Assay.

The reverse phase HPLC assay allowed both serum phenylacetate andphenylacetylglutamine concentrations to be determined simultaneouslyfrom the same sample (see FIG. 12). The lower limit of detection forboth compounds in serum and urine was 5 μg/ml, based upon asignal-to-noise ratio of 5:1. The interassay CV for serum concentrationswas less than 6% within the range of 40 to 1,000 μg/ml. (Table 14). Thelower limit of detection for glutamine was 0.5 μg/ml, with an interassayCV that did not exceed 7%.

Model Specification and Initial Parameter Estimation.

FIG. 13 shows representative concentration versus time curves forsimultaneously measured serum levels of phenylacetate andpheylacetylglutamine and plasma levels of glutamine following a 150mg/kg bolus dose of sodium phenylacetate. The post-infusion decline inserum phenylacetate concentration over tim eis linear when plotted on anon-logarithmic scale, consistent with saturable elimination kinetics.While useful for demonstrating a zero-order process, the 150 mg/kg boluswas inadequate for parameter estimation insofar as most of thephenylacetate concentrations obtained over the six-hour sampling periodwere above K_(m). In order to generate concentrations both above andbelow K_(m), the bolus was changed to 60 mg/kg i.v. over 30 minutes.Visual inspection of the concentration versus time curves followingthese boluses revealed no evidence of an initial distributive phase,suggesting that a single compartment, open non-linear model should beadequate to describe the drug's pharmacokinetics. Initial estimates(mean±SD) of K_(m) (90±30 μg/ml), V_(max) (26.0±10 mg/kg/hr) and Vd(22.4±6.8 L) were calculated in 13 patients using the Lineweaver-Burkequation. Refinement of these initial parameter estimates by non-linearleast squares fitting of the entire concentration versus time profilefor each bolus dose yielded the following estimates: K_(m) =105.1±44.5μg/ml, V_(max) =24.1±5.2 mg/kg/hr and Vd=19.2±3.3 L. The differencesbetween the two methods of estimation were not statistically different,as measured by the Student's t-test (p=0.89).

Induction of Phenylacetate Clearance.

In some patients treated at dose levels 1 and 2, we observed a tendencythe serum phenylacetate concentration to decrease with time. An exampleof this phenomenon is shown in FIG. 14. Considering the 12 cycles oftherapy delivered at these levels, a comparison of the serum drugconcentration measured on day 2 of CIVI to that observed on day 11demonstrated a statistically significant decline in concentration withtime (Wilcoxon signed rank test, p=0.016). At dose levels 3 and 4,attempts at maintaining targeted serum phenylacetate concentrationsusing adaptive control with feedback led to variable rates of druginfusion over time, which precluded a simple comparison of drugconcentrations at the beginning and end of therapy.

Therefore all cycles of therapy were analyzed at all four dose levelsand compared with the performance of the single compartment non-linearmodel described above with the same model modified to allow V_(max) toincrease with time. The formula used to describe this increase was:

    V.sub.max, (1)=V.sub.max, t=0 ×{1.0+[(IF-1.0)×(1.0-e.sup.IRλt)]}

wherein t is the time elapsed (in hours) since the initiation oftherapy, IF is an induction factor representing the maximum-foldincrease in V_(max) at infinite time and IR is a first order rateconstant (h⁻¹) describing the rate at which V_(max) increases over time.Each cycle of therapy (n=21) was evaluated by comparing the differencein the weighted sum of errors squared generated by non-linearleast-squares-fitting with each model. The significance of thedifference was evaluated using the F test (see statistical methods). In9 of the 21 cycles, allowing V_(max) to increase with time yielded animproved fit (induction parameters, mean±SD:IF=1.87±0.37,IR=0.0028±0.003 h⁻¹, p≦0.035). The Spearman rank correlation method didnot demonstrate a correlation between rate of drug administration andthe need to incorporate the two induction parameters into the model(rank correlation coefficient=0.39, p=0.084). The dose ratesadministered ranged from 450 to 1,850 mg/h.

Review of concomitantly administered medications revealed no associationbetween specific drugs and the occurrence of a time-dependent increasein phenylacetate clearance. In seven patients with primary CNS tumors,treatment with anticonvulsants always antedated the administration ofphenylacetate by months to years.

Mechanisms of Phenylacetate Clearance.

As shown in FIG. 13, phenylacetate underwent rapid conversion tophenylacetylglutamine. In the three patients who received 150 mg/kg ofphenylacetate over 2 hours, the peak serum concentration ofphenylacetylglutamine (mean±SD) was 224±81 μg/ml, 325±72 minutespost-infusion. After 60 mg/kg boluses, the peak serumphenylacetylglutamlne concentration was 104±33 μg/ml at 86±33 min. Theplasma glutamine concentration prior to bolus treatment withphenylacetate was 105±29 μg/ml (mean±SD, n=16), similar to reportedvalues in the literature for normal volunteers. The largest reduction incirculating plasma glutamine levels (46%) was observed in a patientreceiving a 150 mg/kg bolus.

The molar excretion of phenylacetylglutamine was determined from 24 hoururine collections. It accounted for 99±23% (n=18) of the dose ofphenylacetate administered over the same period of time. The recovery ofthe free, non-metabolized drug was only 1.5±2.4% of the totaladministered dose. A strong phenylacetate odor was detectable onpatients' clothes and on examiners' hands after physical examination.This suggests that phenylacetate may also be excreted to some extenttransdermally.

Distribution of Phenylacetate and Phenylacetylglutamine into the CSF.

Clinical circumstances required evaluation of the cerebrospinal fluid intwo patients who had metastatic prostate cancer and were free of CNSmetastases. The first had reached steady-state phenylacetate andphenylacetylglutamine concentrations of 141 and 199 μg/ml, respectively,the corresponding simultaneous CSF concentrations were 74 and 5 μg/ml,respectively. At the time of simultaneous serum and CSF sampling, thesecond patient had been off therapy for 6 hours after having reached aserum concentration of phenylacetate of 1044 μg/ml. Measurements inserum and CSF were 781 versus 863 μg/ml for phenylacetate and 374 versus46 μg/ml for phenylacetylglutamine, respectively.

Clinical Toxicities.

No toxicity was associated with bolus administration of the drug. Thehighest peak serum concentrations were measured after the 150 mg/kgbolus over 2 hours (533±94 μg/ml, mean±SD). Table 15 lists the averageserum phenylacetate concentrations per dose level. Although thoseachieved at dose levels 3 and 4 are close to their target, the largestandard deviations reflect our inability to maintain serumphenylacetate concentrations within the desired range, even when usingadaptive control with feedback.

Drug-related toxicity was clearly related to the serum phenylacetateconcentration. Three episodes of CNS toxicity, limited to confusion andlethargy and often precided by emesis, occurred in patients treated atdose levels 3 and 4. They were associated with drug concentrations of906, 1044 and 1285 μg/ml (mean: 950±300 μg/ml), respectively. Symptomswere completely resolved within 18 hours of terminating the druginfusion in all instances,

Antitumor Activity.

Stabilization of PSA for more than 2 months was noted in 3 of the 9patients with prostate cancer treated at dose levels 2, 3 and 4 (meanphenylacetate concentration: 234±175 μg/ml). A fourth patientexperienced marked improvement in bone pain and was able to substitute anon-steroidal anti-inflammatory drug to his morphine regimen. Onepatient with glioblastoma multiform has had improvement in performancestatus (30% on Karnofsky's scale), intellectual function and expressiveaphasia of greater than 5 months duration. Although no change in thesize of the tumor mass was noted, reduction in peritumoral edema wasdocumented by MRI.

Discussion.

Previous descriptions of the pharmacokinetics of phenylacetate have beenfragmentary. Simell et al. reported the drug to have first orderelimination kinetics with a half-life of 4.2 hours following bolus doseadministration 9270 mg/kg) children [Simell, O., Sipila, I., Rajantie,J., Valle, D. L., and Brusilow, S. W. Waste nitrogen excretion via aminoacid acylation: benzoate and phenylacetate in lysinuric Proteinintolerance. Pediatr. Res., 20:1117-1121, 1986]. The failure torecognize the non-linear nature of phenylacetate pharmacokineticsprobably resulted from the smaller total doses given to these patientscompared to those given in our study. The saturable pharmacokinetics ofphenylacetate are consistent with an enzymatic process and ourcalculations from the 24 hour urinary excretion of phenylacetylglutamineconfirm that this is the major route of elimination. Evidence that drugclearance increases with time was derived from the comparison of druglevels on days 2 and 11 of the CIVI, adding another layer of complexityto the pharmacokinetics of phenylacetate. To explain this phenomenon,the potential role of concomitantly administered medications was firstconsidered, but failed to demonstrate any association. Analysis of a therelationship between an increase in drug clearance with time and therate of drug administration did not reach statistical significance andsuffered from the small number of cycles of therapy available foranalysis. It should also be noted that, relative to the 14 day periodover which it is assessed, V_(max) tended to increase slowly, with anaverage half-time calculated from the induction rate (IR) of 9.6 days.

As expected for such a small molecule, phenylacetate readily penetratesinto the CSF, which may explain the dose-limiting side-effects of thedrug, i.e., nausea, vomiting, sedation and confusion.

The results of Table 15 indicate that attempting to maintain serumphenylacetate concentrations at either 200 or 400 μg/ml using adaptivecontrol with feedback was problematic, with drug concentrations thatoften greatly exceed the level-specific targets. All patients whoexhibited CNS toxicity had serum phenylacetate concentrations in excessof 900 μg/ml. In the average patient, the drug must be infused at a rateequal to 75% of V_(max), in order to maintain a constant serumphenylacetate concentration of 400 μg/ml, which is four times greaterthan K_(m). Thus, the slightest error in the estimation of individualpharmacokinetics or in the rate of drug infusion results in largechanges in drug concentration. Phenylacetate was delivered by CIVI inorder to mimic the preclinical conditions that had demonstratedantitumor activity, namely, continuous exposure to concentrations above475 μg/ml for at least two weeks. Unfortunately, such concentrationscannot be practically maintained.

An alternative strategy is to deliver the drug by repeated shortinfusions. Our limited experience with the 150 mg/kg i.v. bolusessuggests that serum phenylacetate concentrations occurring transientlyabove 500 μg/ml are well tolerated. In addition, the time intervalbetween infusions allows some drug washout to occur, thereby minimizingdrug accumulation. A simulated regimen of 200 mg/kg q 12 h (1 hourinfusion) is presented in FIG. 16. The simulation assumes that thepharmacokinetic parameters determined from our 17 patients arerepresentative of the cancer population at large and that V_(max) doesnot change with time. It predicts that a wide range of peak drugconcentrations will be present. However, it is possible that these wouldbe sufficiently transient so as not to produce CNS toxicity and thetroughs not so prolonged as to abrogate the drug's antitumor activity.

                  TABLE 14                                                        ______________________________________                                        PA Standard Curve Assay Variability                                           PA        CV           PAG      CV                                            (μg/ml)                                                                              (%)          (μ/ml)                                                                              (%)                                           ______________________________________                                         40       2.6           40      4.6                                            400      1.7           400     4.3                                           1000      3.4          1000     3.1                                           ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        PA and PAG Concentrations                                                     Per Dose Level During CIVI                                                                            PA.sup.a  PAG.sup.a                                   Dose Level PA dose level                                                                              (μg/ml)                                                                              (μg/ml)                                  ______________________________________                                        1          150 mg/kg/d  49 ± 19                                                                               90 ± 34                                 2          250 mg/kg/d  104 ± 40                                                                             150 ± 63                                 3          200 μg/ml 178 ± 85                                                                             188 ± 55                                 4          400 μg/ml 397 ± 244                                                                            306 ± 51                                 ______________________________________                                         .sup.a mean ± SD                                                      

EXAMPLE 16

Effect of NaPA on differentiation of human neuroblastoma cells.

The ability of NaPA to promote the differentiation of humanneuroblastoma cells was studied, both alone and in combination withretinoic acid (RA), a known inducer of neuroblastoma differentiation andmaturation. In the LA-N-5 cell line, phenylacetate stimulated thedifferentiation of human neuroblastoma cells as evidenced bydose-dependent inhibition of cell proliferation, neurite outgrowth,increase acetylcholinesterase activity, and reduction of N-myc proteinlevels. Furthermore, NaPA and RA synergized in inducing LA-N-5differentiation in that combination treatment resulted in completecessation of cell growth along with morphologic and biochemical changesindicative of the loss of malignant properties. The combined effectsrepresent a strong differentiation response in neuroblastoma cells, bothas to number of responding cells and maturational level achieved.Transient transfection of LA-N-5 cells with a variety of CAT reportergene plasmids including constructs containing thyroid and RA responsiveregulatory elements have suggested that the pathways of action of NaPAand RA may intersect at the nuclear level through activation of commonresponse elements. The synergistic effects, thus, may be mediated by theability of NaPA to modulate the RA differentiation pathway so as toresult in altered transactivation of RA responsive regulatory elementsin relevant target genes. These in vitro antineoplastic effects wereobserved under drug concentrations achievable in humans withoutsignificant toxicities.

SECTION B: PHENYLACETATE AND ITS DERIVATIVES IN THE TREATMENT ANDPREVENTION OF AIDS

The etiology of human acquired immunodeficiency syndrome (AIDS) has beenlinked to the human immunodeficiency virus (HIV), which is capable ofselective infection and suppression of the host immune system. Thisimmune defect renders the human body susceptible to opportunisticinfections and cancer development, which are ultimately fatal. Thespread of HIV throughout the world is rapid, with no effectivetherapeutics on hand. It is suggested that NaPA, a nontoxic naturalcompound capable of glutamine depletion in vivo, can be used in thetreatment and prevention of AIDS.

HIV is a retrovirus. The production of retroviruses is dependent ontranscriptional activation by the long terminal repeat (LTR) element,and the availability of glutamine (Gln) for translational control.Experimental data obtained with chronically infected cultured cells andanimal models indicate that virus replication is specifically inhibitedin cells starved for glutamine, but not in those starved for other aminoacids (Gloger and Panet (1986); (J. Gen. Virol. 67:2207-2213) Robertsand McGregor, (1991), (J. Gen. Virol 72:2199-305). The results could notbe attributed to either an effect on cell cycle or a general inhibitionof protein synthesis.

The reason why glutamine depletion leads to virus suppression can beexplained as follows. Replication competent murine retroviruses containan amber termination codon at the junction of gag and pol genes, whichcan be recognized by amber suppressor tRNA^(Gln). Glutamine is thusessential for the readthrough of viral mRNA transcripts [Yoshinaka etal. (1985); PNAS 82:1618-1622]. Reduction in glutamine concentrationsdisrupts viral mRNA translational readthrough and protein synthesis,with subsequent inhibition of viral assembly and secondary spread.Although human retroviruses are somewhat different from the murineviruses studied, it has been shown that reduction in the levels of ambersuppressor tRNA^(Gln) in human cells infected with HIV causes asignificant reduction in the synthesis of vital proteins [Muller et al.Air Research and Human Retroviruses 4:279-286 (1988)]. Such data suggestthat agents which can lower glutamine levels in humans are likely tobenefit patients infected with HIV. NaPA may be such an agent, since itis known to conjugate to glutamine in humans with subsequent renewedexcretion of phenylacetylglutamine. Since NaPA also possesses antitumoractivities, the drug is likely to affect Kaposi's sarcomas, the tumorsfound in as many as 30% of all AIDS patients, as well as lymphomasassociated with AIDS.

EXAMPLE 17

NaPA for treatment of AIDS related disorders

Evidence from experimental model systems in support of the abovehypotheses includes:

(a) Preliminary findings with cultured cells indicate that NaPA caninhibit expression of genes controlled by the retroviral LTR; (b) Whileanimal studies have been hindered by the fact that glutamine depletionby NaPA is limited to humans and higher primates, an acceptable animalmodel (other than primates) involves rodents treated with glutaminase.The expression of retroviral genes is under the control of the longterminal repeat (LTR) element; inhibition of LTR would preventtranscription and synthesis of viral proteins. To examine the effect ofNaPA on the retroviral LTR, V7T fibrosarcoma cells carrying anLTR-dependent Ha-ras oncogene were used as a model. Results of Northernblot analysis showed markedly reduced levels of the ras RNAtranscription in cells treated with NaPA compared to RNA transcriptionlevels in untreated control cells. The results cannot be explained by ageneral effect on gene expression, as indicated by the increasedexpression of the cellular genes collagen and 2'-5' oligo adenylatesynthetase (2-5 ASyn). The latter are of particular interest sincecollagen is a marker of fibroblast differentiation, and 2-5 ASyn isassociated with growth control. Taken together, the data indicate theNaPA suppressed the activity of the retroviral LTR, while restoringgrowth control and differentiation to the host cells. Similarlydesirable changes might occur in HIV-infected monocytes and T4lymphocytes following systemic treatment of afflicted patients withNaPA. Glutaminase is a bacterial enzyme that causes reduction ofextracellular (and presumably intracellular) glutamine concentrations.Glutaminase treatment of viremic mice infected with Rouscher murineleukemia virus (RLV) inhibited retroviral replication and thedevelopment of splenomegaly, and significantly increased animal survival[Roberts and McGregor J. Gen. Virology 72:29-305 (1991)]. The efficacyof glutaminase therapy compared favorably with AZT, the drug currentlyused for treatment of AIDS. The results are of particular interest sincethe RLV serves as a model in the search for anti-HIV drugs (Ruprecht etal., 1986). Unfortunately, however, glutamine depletion by glutaminasein vivo is only transient due to development of neutralizing antibodiesto the enzyme. Once this occurs, vital replication can resume,eventually killing the host. NaPA, unlike the bacterial glutaminase, isa natural component of the human body, and thus is less likely to inducethe production of neutralizing antibodies; (c) There is clinicalevidence for sustained reduction by NaPA of plasma glutamineconcentrations. NaPA is currently being used for treatment ofhyperammonemia associated with inborn disorders of urea metabolism.Clinical experience indicates that long-term treatment with NaPAeffectively reduces glutamine levels. Such treatment is nontoxic andwell tolerated even by newborns. In conclusion, NaPA might benefitpatients with HIV infection. NaPA could inhibit viral replicationthrough (among other mechanisms) inhibition of LTR and depletion ofglutamine, the amino acid required for appropriate processing of vitalproteins. If NaPA proves to have anti-HIV activities in humans, it couldbe used to prevent disease progression in asymptomatic HIV-positiveindividuals. The lack of toxicity, easy oral administration andrelatively low cost uniquely qualify NaPA as a chemopreventive drug. Infact, the drug is so well tolerated by humans that treatment can startjust a few hours after birth. In addition, NaPA could be used (alone orin combination with other drugs) in treatment of AIDS-associateddisorders including opportunistic infections, HIV encephalopathy, andneoplasia.

SECTION C: INDUCTION OF FETAL HEMOGLOBIN SYNTHESIS IN β-CHAINHEMOGLOBINOPATHY BY PHENYLACETATE AND ITS DERIVATIVES

There is considerable interest in identifying nontoxic therapeuticagents for treatment of severe β-chain hemoglobinopathies. Employing thehuman leukemic K562 cell line as a model, we have explored the cellularresponses to NaPA, an amino acid derivative essentially nontoxic tohumans. Treatment of cultures with pharmacologically attainableconcentrations of NaPA resulted in time- and dose-dependent inhibitionof cell proliferation and caused an increase in hemoglobin production.Molecular analysis revealed accumulation of the fetal form of hemoglobin(HbF), which was associated with elevated steady-state levels of gammaglobin mRNA. All NaPA effects reversed upon cessation of treatment.Interestingly, addition of NaPA to other antitumor agents of clinicalinterest, i.e., 5-azacytidine and hydroxyurea, resulted insuperinduction of HbF biosynthesis. The results suggest that NaPA, anagent known to be well tolerated by newborns, could be used alone or incombination with other drugs for long-term treatment of some inbornblood disorders.

The pathophysiology of inherited blood disorders such as sickle cellanemia and severe β-thalassemias is based on genetic abnormalities inthe β-globin gene which result in deficient or absent β-globinsynthesis. The latter prevents the production of hemoglobin and resultsin ineffective red blood cell production and circulation. Recent dataindicate that pharmacological manipulation of the kinetics of cellgrowth and differentiation might have a beneficial effect in patientswith the β-chain hemoglobinopathies, due to the induction of fetalhemoglobin (HbF) synthesis. To date, several antitumor drugs including5-azacytidine (5AzaC), 5-aza-2'-deoxycytidine (5AzadC), hydroxyurea(HU), vinblastine, and arabinosylcytosine (ara-C) have been shown toincrease the production of HbF in experimental models [Dover, Ann NYAcad. Sci. 612:184-190 (199)]. Moreover, there is clinical evidence for5AzaC and HU activity in severe β-thalassemia and sickle cell anemia,respectively. However, concerns regarding toxic and potentialcarcinogenic effects of the prevailing antitumor drugs raise the need toidentify safe alternatives for long-term treatment of the inbornnonmalignant diseases. The accumulation of fetal hemoglobin in adults isthought to be due to changes in the kinetics of erythroiddifferentiation rather than a direct effect on the fetal globin genes.According to this hypothesis, other agents that can inducedifferentiation would also be expected to affect HbF production. Thefocus here is on the efficacy of a novel nontoxic differentiating agent,NaPA.

As discussed in Section A, Applicant's laboratory has found that NaPAcan also affect the maturation (i.e., differentiated state) of variousanimal and human cell types. The drug caused growth arrest and reversalof malignant properties in a variety of in vitro tumor models includingcell lines established from adenocarcinomas of the prostate and lung,malignant melanomas, and astrocytomas. Moreover, NaPA treatment wasassociated with adipocyte conversion in premalignant mesenchymal C3H10T1/2 cells, and granulocyte differentiation in promyelocytic leukemiaHL-60 cultures. Studies indicated that NaPA, in contrast to thechemotherapeutic differentiating drugs 5AzaC and 5AzadC, may be free ofadverse effects such as cytotoxicity and tumor progression.

Indeed, NaPA is well tolerated by humans as indicated by the vastclinical experience with NaPA in the treatment of hyperammonemia ininfants with inborn errors of ureagenesis. The clinical experienceindicates that acute or long-term treatment with high doses of NaPA isessentially free of adverse effects. The lack of toxicity and theability to induced cellular differentiation prompted Applicant toexamine the effect of NaPA on HbF expression.

EXAMPLE 18

K562 cells--induction of HbF by treatment with NaPA

The experimental system involved the human leukemic K562 cells, whichcarry a nonfunctional β-globin gene, but produce low levels of the fetalgamma globin and of HbF. The K562 cell line was originally establishedfrom a patient with chronic myelogenous leukemia in the blast cellstransformation, and has since been extensively utilized as a model instudies of erythroid differentiation and regulation of the gamma globingene expression. Applicant has shown that pharmacologically attainableconcentrations of NaPA can promote HbF biosynthesis in the humanleukemic cells, and can cause superinduction when combined with theother chemotherapeutic agents of interest, 5AzaC and Cell Culture andreagents.

The human leukemia K562 cells were maintained in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum (Gibco), 50 U/mlpenicillin, 50 μg/ml streptomycin, and 2 mM L-glutamine unless otherwiseindicated. The suspension cultures were kept in exponential growth phaseby diluting every 3-5 days with fresh medium, and cell viability wasdetermined by trypan blue exclusion. Phenylacetic acid,4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid,2,5-dihydroxyphenylacetic acid (Sigma, St. Louis, Mo.) and PAG (a giftfrom L. Trombetta, Houston, Tex.) were dissolved in distilled water, andbrought to pH 7.0 by the addition of NaOH, DON, adivicin, 5AzadC, 5AzaC,and HU (Sigma) were also dissolved in distilled water. All drug stocksolutions were stored in aliquots at -20° C. until used.

Determination of Hemoglobin Production.

K562 cells were seeded at 1×10⁵ cells/ml and treated with the drugs forfour to seven days prior to assay. Qualitative estimation of hemoglobinproduction was determined by benzidine staining of intact cells insuspension. The hemoglobin concentration within cells was determined bythe protein absorption at 414 nm. Briefly, 1×10⁷ cells were lysed in 1ml of lysing buffer (0.12% Tris pH 7.4, 0.8% NaCl, 0.03% Mg-acetate, and0.5% Np-40), vortexed and incubated on ice for 15 minutes. The lysateswere then centrifuged for 15 minutes at 1500 rpm at 4° C., and theabsorption of the supernatant monitored between 350 nm and 650 nm usingBeckman Du-7 scanning spectrophotometer. The hemoglobin was quantitatedusing the relationship of 1.0 optical density (OD) at 414 nmcorresponding to 0.13 mg/ml hemoglobin as described before.

Northern Blot Analysis and DNA probes.

Cytoplasmic RNA was prepared from cultures at logarithmic phase ofgrowth and separated on 1% agarose-formaldehyde gels. Gelelectrophoresis, transfer of RNA onto nytran membranes (Schleicher &Schuell), hybridization with radiolabeled DNA probes, andautoradiography (Kodak X-ray film XAR5) were according to establishedprocedures. The probe for gamma globin was a 0.6 Kb EcoRI/HindIIIfragment of the human gamma globin gene. Probes were labeled with [³²P]dCTP (New England Nuclear, Boston, Mass.) using random primed DNAlabeling kit (Boehringer Mannheim, West Germany).

Analysis of HbF Protein Synthesis.

Newly synthesized proteins were labeled with ³⁵ S-methionine and the HbFimmunoprecipitated and analyzed as previously described. Briefly, cells(1×10⁶ per point in 1 ml) were first subjected to 1 hr starvation inmethionine-free medium, then incubated in the presence of 100 uCi/ml of³⁵ S-methionine for 2 hrs. The labeled cells were harvested, washed andlysed in a lysing buffer containing 10 mM phosphate buffer pH 7.4, 1%Triton×100, 0.1% SDS, 0.5% deoxycholate, 100 mM NaCl, 0>1% NAN3, 2 mMPMSF, and 10 μg/ml lenpeptin. 1×10⁷ cpm of TCA precipitable count ofcytoektract was incubated with rabbit anti-human HbF (Pharmacia) andprotein A Sepharose at 4° C., and the immunoprecipitates were separatedby electrophoresis on 12% SDS-polyacrylamide gels.

The Effect of NaPA and Analogues on Cell Growth and Differentiation.

Treatment of the K562 cultures with NaPA resulted in dose dependentinhibition of cell proliferation, with 1.4 mg/ml causing 50% reductionin cell number after four days of treatment (FIG. 17). No toxicity wasobserved with doses as high as 2.0 mg/ml. In addition to the cytostaticeffect, NaPA also induced erythroid differentiation, as indicated by anincrease in the number of benzidine-positive cells (FIG. 17) andconfirmed by quantitative analysis of hemoglobin production (Table 16).Similar treatment with PAG, which is the glutamine conjugated form ofNaPA, had no significant effect on either cell proliferation orhemoglobin accumulation, suggesting that the changes associated withNaPA treatment are specific and not due to alterations in cultureconditions.

The effect of NaPA on cell growth and differentiation could be mimickedby the use of 4-hydroxyphenylacetate (Table 16). This was in markedcontrast to the analogues 3,4-dihydroxyphenylacetate and2,5-dihydroxyphenylacetate, which were highly toxic to the cells (LD50of 60 and 100 μg/ml, respectively), and did not induce differentiation.

Regulation of Fetal Hemoglobin Production by NaPA.

K562 cells normally express low but detectable levels of HbF. Proteinanalysis employing anti-HbF antibodies revealed significantly increasedamounts of HbF in cells treated with NaPA compared to untreatedcontrols, this was associated with elevated steady-state levels of thefetal gamma globin mRNA. The effect of NaPA on HbF production was timeand dose dependent, and apparently reversible upon cessation oftreatment.

Glutamine Starvation and HbF Production.

NaPA treatment of humans can lead to depletion of circulating glutaminedue to conjugation to glutamine and formation of PAG, an enzymaticreaction known to take place in the liver and kidney. The in vivoreduction in plasma glutamine was mimicked in vitro by culturing theK562 cells in the presence of lowered glutamine concentrations. Resultspresented in Table 17 show, in agreement with previous reports, thatglutamine starvation alone can affect the growth rate as well as HbFproduction in the K562 cells. Addition of NaPA to the glutamine-depletedgrowth medium further augmented the cytostatic and differentiatingeffects observed. Therefore, the effect of NaPA on erythroiddifferentiation and HbF production in humans may be even more dramaticthan that observed with the in vitro model, due to depletion ofcirculating glutamine and a direct effect on the erythroid progenitorcells.

Potentiation by NaPA of Erythroid Differentiation induced by OtherChemotherapeutic Drugs.

There is considerable interest in the use of 5AzaC, 5AzadC and HU fortreatment of sickle cell anemia and β-thalassemia; however, the clinicaluse of these drugs is often limited by unacceptable toxicities.Combination treatments with nontoxic differentiating agents like NaPAcould enhance hemoglobin production while minimizing the adverseeffects. Therefore the efficacy of various combinations of NaPA with theother drugs of clinical interest was tested. Results, summarized inTable 18, show that addition of NaPA 800 μg/ml, to low doses of 5AzadCor HU act synergistically to further augment HbF production with notoxic effect to cells. The concentration of HU used in these experimentsis comparable to the plasma HU levels measured in sickle cell anemiapatients following an oral administration of 25 mg/kg [(Goldberg et al.New England J Med 323:366-372 (1990)]. As to NaPA, pharmacokineticsstudies in children with urea cycle disorders indicate that plasmalevels of approximately 800 μg/ml can be obtained by infusion with300-500 mg/kg/day, a treatment well tolerated even by newborns.

Discussion.

Chemotherapeutic agents selected for their low cytotoxic/mutagenicpotential can be used for induction of fetal hemoglobin in patients withcongenital severe anemias such as sickle cell and β-thalassemia. Drugtoxicity is an important consideration in view of overall healthcondition and the variable life-span of patients with these nonmalignantblood disorders. Unfortunately, recombinant human erythropoietin, whichhas proved to be both nontoxic and effective therapy for anemiasassociated with chronic renal disease, is apparently ineffective in thetreatment of sickle cell anemia. The application of other active drugssuch as 5AzadC, HU, vinblastine and ara-C has been hindered by concernsregarding their carcinogenic effects. HU is also difficult to usebecause of the narrow margin between toxicity and the desired effect onincreased HbF production [Dover, et al., Blood 67:735-738 (1986)]. Incontrast, NaPA, shown here to affect HbF production, is so welltolerated by humans that treatment can be initiated just a few hoursafter birth.

Using an in vitro model involving human leukemic K562 cells, it is shownthat NaPA can promote the maturation of early erythroid progenitor cellsthat have an active HbF program. Addition of NaPA to other therapeuticagents currently in clinical use, i.e., 5AzaC, 5AzadC, or HU resulted insuperinduction of HbF synthesis. 5AzaC has been shown to be less toxicand more effective than HU in stimulating HbF production. Moreover,5AzaC, unlike HU, is effective in treatment of both sickle cell anemiaand β-thalassemia. Such data are consistent with the interpretation that5AzaC acts by both perturbation of erythropoiesis and by its effect onDNA methylation. However, while hypomethylation can lead to geneactivation and cell differentiation, it can also promote oncogenesis andthe evolution of cells with metastatic capabilities. Results obtainedwith the K562 erythroid progenitor cells indicate that the therapeuticeffects of NaPA compare favorably with those of 5AzadC, yet NaPA (unlikethe cytosine analog) did not cause tumor progression. Moreover, NaPA wasshown to prevent tumor progression induced by 5AzadC.

The data show that NaPA, used alone or in combination with otherdrugs,is of value in treatment of leukemias are β-chainhemoglobinopathies. In addition to promoting the production of red bloodcells expressing HbF through nontoxic mechanisms, NaPA may also minimizethe adverse effects of other antitumor drugs currently in clinical use.

                  TABLE 16                                                        ______________________________________                                        HbF Accumulation in Treated K562 Cells                                        Benzidine                                                                              Positive Cells  HbF production                                       Treatment          fold              fold                                     (mg/ml)  (%)       increase  (pg/cell)                                                                             increase                                 ______________________________________                                        None     2.2 ± 0.8                                                                            1         0.35 ± 0.06                                                                        1                                        NaPA                                                                          0.4      2.7 ± 0.2                                                                            1.2       0.49 ± 0.02                                                                        1.4                                      0.8      7.0 ± 0.3                                                                            3.2       1.15 ± 0.20                                                                        3.3                                      1.6      14.6 ± 0.2                                                                           6.6       2.40 ± 0.16                                                                        6.8                                      4HP 1.6  14.2 ± 0.5                                                                           6.45      ND                                               PAG 2.6  2.1 ± 0.5                                                                            0.95      0.37 ± 0.03                                                                        1.06                                     ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        Glutamine Starvation and HbF Production                                                HbF (g/cell)                                                                    Gln starvation                                                     Gln (mM)   alone       Plus NaPA (0.8 mg/ml)                                  ______________________________________                                        2.0        0.39 ± 0.04                                                                             1.0 ± 0.06                                         0.5        0.56 ± 0.01                                                                            1.15 ± 0.01                                         0.2.sup.a  1.17 ± 0.12                                                                            1.75 ± 0.22                                         0.1.sup.a  1.86 ± 0.40                                                                            2.22 ± 0.20                                         ______________________________________                                         .sup.a The concentration of NaPA used in this study (0.8 mg/ml) is            pharmacologically attainable without toxicity. In children such a             treatment is expected to cause a drop in circulating glutamine plasma         levels to 0.1-0.2 mm. The results presented above indicate that under suc     conditions HbF production increases 4.5-5.7 fold compared to controls. We     propose therefore that the effect of NaPA in children might be more           dramatic than that seen under routine culture conditions (i.e., cell          growth in medium with 2 mM Gln).                                         

                  TABLE 18                                                        ______________________________________                                        Potentiation by NaPA of                                                       HU's Therapeutic Effect                                                       Treatment            HbF (pg/cell)                                            ______________________________________                                        None                 0.39 ± 0.04                                           NaPA (0.8 mg/ml)     1.64 ± 0.07                                           HU (50 uM)           1.00 ± 0.03                                           HU (50 uM) + NaPA    5.91 ± 0.6.sup.b                                      HU (100 uM)          2.12 ± 0.04                                           HU (100 uM) + NaPA   6.71 ± 0.05.sup.b                                     ______________________________________                                         .sup.a To mimic the effect of NaPA in vivo, treatments involving NaPA wer     performed in medium supplemented with 0.2 mM Gln (see explanation to Tabl     17). Control untreated cells and those treated with HU or 5AzadC alone        were maintained in growth medium with 2 mM Gln.                               .sup.b The results indicate that NaPA and HU act synergistically to induc     HbF Production int he erythroid progenitor cells                              Note: Similar results have been obtained for the combination NaPA 0.8         mg/ml and 5AzadC 0.3 uM.                                                 

EXAMPLE 19

HbF induction in nonmalignant and malignant cells

General ability of NaPA and its derivatives to induce production of HbF.

The ability of oral administration of sodium 4-phenylbutyrate toincrease fetal hemoglobin production was assayed. To do so, thepercentage of red cells containing fetal hemoglobin (F cells) wasmeasured by flow-cytometric single-cell immunofluorescent assays in 15patients (7 females and 8 males) with hereditary urea-cycle disorderswho had received sodium 4-phenylbutyrate therapy for 5 to 65 months. Indetermining the differences in low levels of fetal hemoglobin in personswithout anemia, the measurement of the percentage of F cells is moreprecise than conventional measurements of fetal hemoglobin as apercentage of total hemoglobin. The mean percentage of F cells wassignificantly higher in the patients than in normal subjects:

    ______________________________________                                                            Dose of                                                   Patient    Age      Phenylbutyrate                                                                            F Cells*                                      No.        yr       g/kg/day    %                                             ______________________________________                                        1          29       0.30        9.4                                           2          11       0.67        20.4                                          3          6        0.62        0.5                                           4          5        0.48        6.5                                           5          2        0.58        22.7                                          6          13       0.46        7.7                                           7          2        0.38        11.8                                          8          11       0.41        1.9                                           9          6        0.27        1.9                                           10         5        0.62        2.3                                           11         6        0.65        21.1                                          12         21       0.29        1.7                                           13         3        0.47        7.6                                           14         6        0.64        40.5                                          15         2        0.63        29.7                                          Patients,           --           12.4 ± 3.1**                              mean ± SE                                                                  Normal subjects,    --          3.1 ± 0.2                                  mean ± SE                                                                  ______________________________________                                         *F cells were measured with a flowcytometric technique that counts the        percentage of F cells in a total of 10,000 red cells. The difference          between repeated measurements was less than 10 percent.                       **P = 0.005 by the KolmogorovSmirnov twosample test for the comparison of     the Fcell values in the 15 patients with ureacycle disorders and the          values in 293 normal adults. The percentage of F cells reaches the range      of values found in normal adults at about two years of age.              

EXAMPLE 20

In vitro study of sickle cell and beta-thalassemia responses toNaPA/NaPB

An in vitro study was conducted on cells derived from patients withhomozygous sickle cell disease or B-thalassemia who had been admitted tothe Clinical Center of the National Institutes of Health (NIH) forroutine evaluation, or normal blood donors from the Department ofTransfusion Medicine (NIH). Approximately 20-25 ml of blood was obtainedfor erythroid cell cultures. Diagnosis of SS or B-thal was made on thebasis of: (1) hemoglobin electrophoresis on alkaline cellulose acetateand on acid citrate sugar; (2) peripheral blood examination; andoccasionally (3) DNA and RNA analysis of bone marrow aspirates. Whenpossible, diagnosis was confirmed by family studies. Routine hematologicprofiles were performed on a Coulter Model S.

Peripheral blood mononuclear cells were isolated by centrifugation on agradient of Ficoll-Hypaque and cultured for 7 days (phase I) inalpha-minimal essential medium supplemented with 10% fetal calf serum(FCS) (both from GIBCO, Grand Island, N.Y.), 1 μg/ml cyclosporin A(Sandoz, Basel, Switzerland) and 10% conditioned medium collected frombladder carcinoma 5637 cultures (Myers C. D., Katz F. E., Joshi G,Millar J. L.: A cell line secreting stimulating factors for CFU-GEMMculture. Blood 64:152, 1984). In phase II, the non-adherent cells wererecultured in alpha-medium supplemented with 30% FCS, 1% deionizedbovine serum albumin, 1×10⁵ M 2-mercaptoethanol, 1.5 mM glutamine(unless otherwise indicated), 1×10⁶ M dexamethasone, and 1 U/ml humanrecombinant Epo (Ortho Pharmaceutical Co., Raritan, N.J.). Thesecultures yielded up to 10⁶ erythroid cells per milliliter of blood. Cellviability was determined by Trypan Blue exclusion. Phenylacetic acid,4-phenylbutyric acid, p-hydroxyphenylcetic acid, p-chlorophenylaceticacid, and butyric acid (Sigma, St. Louis, Mo.) were dissolved indistilled water and brought to pH 7.0 by the addition of NaOH.5-Azacytidine and hydroxyurease was obtained from Sigma, and PAG wasobtained from S. Brusilow (Johns Hopkins, Baltimore, Md.).

Differentiation was assessed morphologically by preparing cytocentrifugeslides stained with alkaline benzidine and Giemsa. The number ofHb-containing cells was determined using the benzidine-HCl procedure(Orkin S. H., Harosi F. L., Leder P.: Differentiation of erythroleukemiccells and their somatic hybrids. Proc Natl. Acad. Sci U.S.A. 72:98,1975). Hbs were characterized and quantitated by cation exchange highperformance liquid chromatography (HPLC) of cell lysates as previouslydescribed (Huisman TH: Separation of hemoglobins and hemoglobin chainsby high performance liquid chromatography. J Chromatography 418:277,1987). Total Hb in lysates prepared from a known number of Hb-containing(benzidine-positive) cells was measured using either thetetramethylbenzidine procedure (Sigma kit, Catalog No. 527) or by cationexchange HPLC (measuring total area under chromatogram). Standard Hbsolutions (Isolab, Inc., Akron, Ohio) were used for reference. Meancellular Hb (MCH) was calculated by dividing the total Hb content of thelysate by the number of benzidine-positive cells.

Cytoplasmic RNA was separated on 1% agarose-formaldehyde gels. RNAisolation, gel electrophoresis, transfer onto Nytran membranes(Schleicher & Schuell, Inc., Keene, N.H.), hybridization withradiolabeled DNA probes, and autoradiography (Kodak X-ray film XARS)were described [Samid D., Yeh A., Presanna P.: Induction of erythroiddifferntiation and fetal hemoglobin production in human leukemic cellstreated with phenylacetate. Blood, 80:1576, 1992]. The human globin cDNAprobes included JW101 (alpha), JW102 (beta), and a 0.6 kb EcoRI/HindIIIfragment of the 3' end of human G-gamma-globin gene. Probes were labeledwith [³² P]dCTP (New England Nuclear, Boston, Mass.) using a randomprimed DNA labeling kit (Boehringer, Mannheim, Germany). Results.Addition of NaPA or NaPB to phase II erythroid cultures resulted inreduced cell proliferation with no apparent change in cell viability.Cytostatis was associated with a decline in total Hb produced perculture; however, both Hb content per cell (MCH) and the proportion ofHbF (% HbF) increased upon treatment (FIG. 18). The extent of changesobserved was dose- and time-dependent: the earlier the drugs were addedduring the second phase of growth, the higher was the increase in % HbF,however, cell yields were proportionately decreased. For example,addition of 5 mM NaPA to normal precursors on day 2 caused approximately90% decrease in cell number along with a 12-fold increase in % HbF, adetermined on day 13. When treatment was initiated on day 67, cellnumber decreased on by 60% compared to controls, and % HbF increased3.3-fold. In order to obtain sufficient cells for further analysis,subsequent experiments involved the addition of drugs on days 6-7, andcells were harvested on day 13. Under these conditions, results werereproduced in cultures derived from 6 normal donors as well as 4patients with sickle cell anemia and 4 patients with B-thal. NaPA (5 mM)and NaPB (2.5 mM) caused a significant increase in both MCH(38-100%) andthe proportion of HbF produced. In the case of homozygous SS patients, %HbF was elevated 2.0-4.1 fold (mean 3.0) by 4 mM NaPA, and 3.2-5.6 fold(mean 4.0) by 2.5 mM NaPB. The latter was associated with a 12±3%decrease in HbS levels, with no change in HbA₂ (FIG. 19).

As in K562 cells, increased HbF production by NaPA or NaPB in primarycultures of normal or SS cells appears to be due to pre-translationalregulation of gamma-globin expression. Northern blot analysis showeddose-dependent increase (up to 5 fold) in the steady-state levels ofgamma globin mRNA, accompanied by a slight decrease (less than two fold)in the amounts of beta globin transcripts. There was no change in alphaglobin expression.

PAG, the end-metabolite of both NaPB and NaPA, is formed byphenylacetate conjugation to glutamine with subsequent excretion in theurine. PAG was found to be inactive on erythroid proliferation and HbFaccumulation. Glutamine starvation of the non-malignant erythroid cellshad no effect on either cell growth or HbF production, nor did itenhance the efficacy of NaPA.

The effect of NaPA with other drugs was also assayed. When used alone incultures derived from normal donors (HbF base levels of 0.8-2.0%), NaPA(5 mM) and hydroxyurease (0.05 mM) increase % HbF by 3.5 and 2.0-fold,respectively; the combination of the two resulted in a 4.7-fold increasein HbF. NaPA also augmented HbF stimulation by butyrate (0.5 mM) (from3.1 to 7.15-fold), and of 5-Azacytidine (2 uM) (from 2.5 to 6.6-fold).These results indicate that NaPA when added to suboptimal, non-toxicdoses of other drugs, can potentlate HbF production with significantcytostasis and no signficant change in cell viability.

As exemplified below in Table 20, combination treatment comprisingadministration of NaPA (or a pharmaceutically acceptable derivative ofphenylacetic acid) simultaneously with hemin, a known stimulator of HbFproduction, synergistically increases the induction of erythroiddifferentiation, as indicated by the increase in the number of benzidinepositive cells, and HbF production. In K562 cells, the range of increasein the production of HbF with this combination treatment varied from 1.5to 5 times that produced by treatment with 10 mM PA alone. Further,treatment with NaPB in combination with hemin also resulted in classicalsynergism. Similar results were also obtained with PB in non-malignanterythroid progenator primary cells. In all cases, treatment with bothdrugs was maintained for 4-6 days prior to measurement of HbF.

                  TABLE 20                                                        ______________________________________                                        STIMULATION OF HbF BY NaPA IN COMBINATION                                     WITH HEMIN - K562 MODEL                                                       R.sub.x      % Benzidine pos.                                                                           Hb pg/cell                                                                              Viability                                 ______________________________________                                        CONTROL      >0.01        0.26      97                                        NaPA (10 mM) 1.6-3.1      0.91      96                                        NaPA (10 mM) + H                                                                           25.4-32.6    4.03      92                                        NaPA (5 mM) + H                                                                            12.6         2.34      99                                        NaPA (2.5 mM) + H                                                                          8.1          1.95      94                                        HEMIN (20 μM)                                                                           2.9          1.04      98                                        CONTROL      2.1          0.65      97                                        NaPA (2.5 mM)                                                                              2.6          0.91      97                                        NaPA (5 mM)  7.7          1.04      97                                        NaPA (10 mM) 14.3         nd        96                                        HEMIN (20 μM)                                                                           13.8         2.34      nd                                        NaPA (5 mM) + H                                                                            42.3         5.2       97                                        ______________________________________                                    

SECTION D: USE OF PHENYLACETIC ACID AND ITS DERIVATIVES IN WOUND HEALING

Growth factors, including TGF-α, play a critical role in wound healingand repair processes. Wound healing is a localized process that involvesinflammation, wound cell migration and mitosis, neovascularization, andregeneration of the extracellular matrix. Recent data suggest the actionof wound cells may be regulated by local production of peptide growthfactors which influence wound cells through autocrine and paracrinemechanisms (Schultz et al., J. Cell Biochem. 45(4):346 (1991); Schultzet al., Acta Ophthalmol. Suppl.(Copenh), 202:60 (1992)). Two peptidegrowth factors which may play important roles in normal wound healing intissues such as skin, cornea, and the gastrointestinal tract are thestructurally related epidermal growth factor (EGF) and TGF-α, whosereceptors are expressed by many types of cells including skinkeratinocytes, fibroblasts, vascular endothelial cells, and epithelialcells of the gastrointestinal tract. EGF or TGF-α is synthesized byseveral cells involved in wound healing, including platelets,keratinocytes, activated macrophages and corneal epithelial cells.Healing of a variety of wounds in animals and patients, such asepidermal regeneration of partial thickness burns, dermatome wounds,gastroduodenal ulcers and epithelial injuries to the ocular surface, isenhanced by exogenous treatment with EGF or TGF-α. TGF-α, which is apotent inducer of lysyl oxidase mRNA levels in cultures of human scleralfibroblasts, may be primarily responsible for inducing synthesis ofextracellular matrix components after an injury. Furthermore, TGF-α isknown to promote anglogenesis.

The lack of adequate stimulation of growth factors contributes to thenonhealing conditions of many chronic wounds. Poorly healing conditionscould markedly benefit from either addition of exogenous TGF-α orstimulation of effector cells to produce TGF-α and related growthfactors. It has now been discovered that PA and PB (or apharmaceutically acceptable derivative) are capable of stimulatingproduction of TGF-α in cells of melanocytic origin; astrocytic lineage(glioblastoma cells); and several normal human epithelial cell types,including keratinocytes (FIG. 20), which are involved in wound healing.Further, treatment with PA and PB enhances collagen-α type 1 expression.Introduction TGF-α mRNA expression upon treatment with NaPA and NaPB inhuman melanoma cells was observed; expression of TGF-α was confirmedfollowing protein analysis. FIG. 20 shows the increased production ofthe TGF-α protein in human keratinocytes upon exposure to NaPA and NaPB.This increased production of TGF-α is maintained for a few days afterwhich the levels return to approximately pretreatment levels. Asdiscussed below and in FIG. 21, further support for the use thesecompounds in treating wounds may be found in the enhanced expression ofICAM-1, which is a cellular adhesion molecule/surface antigen, followingtreatment with NaPB.

Thus, the instant invention provides a method for stimulating theproduction of TGF-α in cells. Further, wound healing in a human oranimal can be enhanced by treatment with a therapeutic amount ofphenylacetic acid or a derivative of phenylacetic acid such as NaPA orNaPB, which stimulates the in-situ production of TGF-α. For instance,surface wounds can be treated by topically applying PA, PB or aderivative of either PA or PB to the skin surface, such as in a creamformulation. Likewise, ocular injuries can be treated by application ofa PA or PB (or PA/PB derivative) formulation, such as eye drops, to thecornea. Similarly, internal injuries, such as injuries to thegastrointestinal tract, can be treated by administration of oralformulations. Vaginal or anal injuries can also be treated, such as witha suppository containing pharmaceutically effective amounts of PAA or aderivative. The PA/PB or derivative formulations can be administeredcontinuously or, preferably, intermittently, such as one or more dosesin daily, weekly or monthly courses. For example topical administrationonce or twice a day of a composition containing from 0.1 to 10 mM PA,preferably 0.1 to 5.0 mM PA or from 0.1 to 5 mM PB, preferably 0.1 to2.5 mM PB over the course of a week adequately stimulates wound repair.From the information contained herein, dosage concentrations and amountsfor the various administration vehicles can be easily determined. Forinstance, a topical treatment, such as a cream containing PB, typicallywill contain approximately 0.5 to 3.0 mM PB or an equipotent (byequipotent it is meant that dosage may be varied among the differentphenylacetic acid derivatives so as to achieve the equivalent effect onthe subject) dose of a phenylacetic acid derivative. For instance, andwithout limitation, approximately one-half as much PB in a dose isneeded to equal the potency of a similarly indicated PA dose.

SECTION E: USE OF PHENYLACETIC ACID OR ITS DERIVATIVES IN TREATMENT OFDISEASES ASSOCIATED WITH INTERLEUKIN-6

Interleukin-6 (IL-6), which can be produced by monocytes andkeratinocytes upon stimulation, is a pleiotropic cytokine that plays acentral role in defense mechanisms, including the immune response, acutephase reaction and hematopoiesis. Activation of mature B cells can betriggered by antigen in the fluid phase. When antigen binds to cellmembrane IgM in the presence of IL-1 and IL-6, mature virgin B cellsdifferentiate and switch isotypes to IgG, IgA or IgE. Abnormalexpression of the IL-6 gene has been suggested to be involved in thepathogenesis and/or symptoms of a variety of diseases, including (1)non-malignant disorders associated with abnormal differentiationprograms, autoimmunity and inflammatory processes, e.g., rheumatoidarthritis, Castleman's disease, mesangial proliferation,glomerulonephritis, uveitis, sepsis, autoimmune diseases such as lupus,inflammatory bowel, type I diabetes, vasculitis, and several skindisorders of cell differentiation such as psoriasis and hyperkeratosis;(2) viral diseases such as AIDS and associated neoplasms, e.g., Kaposi'sSarcoma and lymphomas; and (3) other neoplasms, e.g., multiple myeloma,renal carcinoma, Lennert's T-cell lymphoma and plasma cell neoplasms.For instance, significantly increased IL-6 mRNA levels in lesionalpsoriatic tissue relative to normal tissue and elevated amounts of IL-6in sera and peripheral blood mononuclear cells of psoriatics compared tosamples from atopics or healthy controls have been found (Elder et al.,Arch. Dermatol. Res., 284(6):324 (1992); Neuner et al., J. Invest.Dermatol., 97(1):27 (1991)).

It has now been discovered that phenylacetic acid or a derivative ofphenylacetic acid, such as NaPA or NaPB, can inhibit the expression ofIL-6. For instance, PA inhibits IL-1-induced IL-6 expression in coloncarcinoma cells. This reduction in RNA is confirmed by reduction in IL-6protein. Thus, PA, PB and their derivatives can be used in the treatmentof diseases involved with the abnormal overexpression of IL-6.

For instance, treatment twice daily by topical application of either 2mM NaPB in a mineral oil-based cream or 2 mM napthylacetate and VitaminB₁ in a mineral oil-based cream directly onto the patient's psoriaticlesions resulted in disappearance of the lesions within a week. Similartreatment of a patient with a severe case of psoriasis resulted in thepsoriatic lesions resolving in approximately 1-3 weeks. Obviously, themode of administration and amount of drug can vary depending upon theIL-6-related disease being treated in order to target the drug to thecells in which reduction of IL-6 expression is desired. For example,injection of a 0.1 mM-5 mM PB solution or an equipotent solutioncontaining a pharmaceutically acceptable phenylacetic acid derivativeinto the joint region may be appropriate for treatment rheumatoidarthritis whereas other diseases may be more appropriately treated bytopical, intravenous or oral delivery. Treatment can be by eithercontinuous or discontinuous treatment, but cessation of the drug,particularly PB, may be accomplished by ramping down the dosage amountsto prevent an overreaction to the cessation of treatment with the drug.Additionally, diseases involving the abnormal overexpression of IL-6 canbe treated by administration of an effective amount of phenylacetic acidor a phenylacetic acid derivative, particularly PA or PB, in combinationwith an effective amount of an anti-inflammatory agent, includingvarious vitamins such as vitamin B₁, non-steroidal inflammatory agentsand steroidal anti-inflammatory agents. The anti-inflammatory agent canbe combined with the phenylacetic acid derivative(s) of this inventionin the same dosage form or administered separately by the same ordifferent route as the derivative. An effective amount of theanti-inflammatory agent refers to amounts currently in clinical use forthe specific disease state or less.

SECTION F: USE OF PHENYLACETIC ACID OR ITS DERIVATIVES IN THE TREATMENTOF AIDS-ASSOCIATED CNS DYSFUNCTION

Hallmarks of central nervous system (CNS) disease in AIDS patients areheadaches, fever, subtle cognitive changes, abnormal reflexes andataxia. Dementia and severe sensory and motor dysfunction characterizemore severe disease. Autoimmune-like peripheral neuropathies,cerebrovascular disease and brain tumors are also observed. In AIDSdementia, macrophages and microglial cells of the CNS are thepredominant cell types infected and producing HIV-1. However, it hasbeen proposed that, rather than direct infection by HIV-1, the CNSdisease symptoms are mediated through secretion of viral proteins orviral induction of cytokines that bind to glial cells and neurons, suchas IL-1, TNF-α and IL-6 (Merrill et al., FASEB J., 5(10):1291(1991)).TGF-β is a growth factor which is released by many cell types. Amongother effects, TGF-β is highly chemotactic for macrophages andfibroblasts and stimulates the release of TNF-α, TGF-α and, indirectly,a variety of other modulators from macrophages which have beenimplicated in the initiation of the CNS symptoms of AIDS.

It has now been discovered that phenylacetic acid or a derivative ofphenylacetic acid, such as NaPA or NaPB, can inhibit the production ofTGF-β2. Because TGF-β2 is an immunosuppresive factor, this inhibitionresults in a general improvement of the patient's immune system. Geneexpression of TGF-β2 in glioblastoma cells was inhibited by both PA andPB. This reduction in RNA leads to reduced TGF-β2 protein synthesis.Thus, PA, PB or their derivatives can be used to inhibit the productionof TGF-β2 in cells, particularly to control or alleviate the CNSsymptoms resulting from HIV infection. As discussed above, thistreatment also inhibits the production of IL-6, further allowing foralleviation of the CNS symptoms. Amounts of drug and/or regimens ofadministration effective for inhibiting TGF-β2 correspond to thoseappropriate for treatment or prevention of cancer as given herein, suchas in SECTION C.

SECTION G: USE OF PHENYLACETIC ACID AND ITS DERIVATIVES TO ENHANCEIMMUNOSURVEILLANCE

Immunosurveillance in an animal such as a human can be enhanced bytreatment with PA, PB or their derivatives. Tumor cells are thought toescape attack by the immune system by at least two means. First, manytumors secrete immune suppressive factors that directly reduce immuneactivity. Additionally, some tumor cells do not express, or have reducedexpression of, appropriate surface antigens that allow the immune systemto identify outlaw cells. However, the compositions of the instantinvention can activate otherwise dormant genes such as fetal globin,perhaps by DNA hypomethylation. Similarly, activation of cancersuppressor genes, dormant antigens and other genes, such as (1) cellularmajor histocompatibility antigens (MHC Class I and II) or other surfaceantigens, such as ICAM-1; (2) tumor antigens such as MAGE-1; and (3)vital latent proteins such as EBV's latent membrane protein (which isimplicated in numerous diseases such as T-cell neoplasms, Burkitt'slymphoma nasopharyngeal carcinoma, and Hodgkin's disease), maycontribute to enhanced immunosurveillance. Thus, neoplastic cells can betreated with PA, PB or their derivatives to provide for expression ofcell surface antigens that increase the effectiveness of the immunesystem by allowing for adequate identification and clearance of thetumor cells by the immune system. Activation of latent viral proteinscould also induce a lytic cycle leading to death of the infected cell.

Evidence that the instant phenylacetic acid or phenylacetic acidderivative compositions can activate dormant genes and enhanceexpression of surface antigens is given by FIG. 21, which shows enhancedexpression of MHC Class I, MHC Class II and the adhesion molecule ICAM-1in melanoma cells that have been treated for 10 days with 2 mM NaPB(e.g., note the shift of the population mean from approximately 50 to200 for MHC class I).

Furthermore, it has now been discovered that PB induces expression ofEBV's latent membrane protein (LMP) in Burkitt's lymphoma cells.Cytoplasmic RNA (20 μg/lane) was isolated from LandisP, RajI and P3HRIBurkitt's lymphoma cell lines, which had been treated with 2 mM PB forfour days, and subjected to Northern blot analysis with a specific LMPprobe. In all three cell lines, a positive reaction was observedcompared to controls (untreated cells), indicating that PB induces theexpression of EBV's latent membrane protein. In Burkitt's lymphoma cellsboth PA and PB cause additional molecular and cellular changes,including cytostasis, decline in myc expression and enhancement ofHLA+1.

Because these surface antigens enhance tumor immunogenicity in vivo,treatment of the animal (human) with PA, PB or their derivatives canenhance the effectiveness of the immune system of the individual. Dosesof approximately 0.5-3.0 mM PB or equipotent doses of pharmaceuticallyacceptable phenylacetic acid derivatives may be useful. This treatmentcan also be combined with conventional immunotherapy treatments and/orantigen targeted, antibody-mediated chemotherapy. While treatmentusually is accomplished by a protocol which allows for substantiallycontinuous treatment, discontinuous or pulsed treatment protocols arealso effective, especially for cells capable of terminallydifferentiating upon treatment with PAA or a PAA derivative. Forinstance, treatment of the melanoma cells given in FIG. 21 for 10 dayswas sufficient to allow continued enhanced expression of the surfaceantigens past this 10 day period.

SECTION H: METHOD OF MONITORING THE DOSAGE LEVEL OF PHENYIACETIC ACID ORITS DERIVATIVES IN A PATIENT AND/OR THE PATIENT RESPONSE TO THESE DRUGS

As discussed above, administration of phenylacetic acid or a derivativeof phenylacetic acid such as NaPA or NaPB to an animal (human) inamounts and over treatment courses as described herein induce a varietyof molecular changes. These molecular traits can be used as biomarkersto either (1) monitor the dosage level of the drug or itsbioavailability in the animal and/or (2) serve as a biomarker of thepatient response to the drug. For instance, as described above,administration of an effective amount of NaPA or NaPB (or theirderivatives) results in a variety of molecular effects, including a)increased levels of fetal hemoglobin in erythrocytes; b) increasedproduction of TGF-α in various cells such as those of melanocyticorigin, astrocytic lineage or epithelial cell types; c) inhibition ofthe production of IL-6; and d) inhibition of the production of TGF-β2.Thus, absolute or relative (before/after treatment) concentrations of aparticular biomarker can be determined in an appropriate cell populationof the individual to allow monitoring of the dosage level orbioavailability of the drug. Further, this concentration can becorrelated or compared with patient responses to develop a patientresponse scale for a desired treatment goal based upon that biomarker.For instance, the increased amount of fetal hemoglobin can be used toindicate the bioavailability of PA or PB for treatment or prevention ofa neoplastic condition as well as indicating the degree of patientresponse to the drug.

SECTION I: THE ACTIVATION OF THE PPAR BY PHENYLACETIC ACID AND ITSDERIVATIVES

Peroxisomes are cellular organelles that contain enzymes which controlthe redox potential of the cell by metabolizing a variety of substratessuch as hydrogen peroxide. Recent advances in this area reveal thatperoxisomes can be proliferated through activation of a nuclear receptorwhich regulates the transcription of specific genes (Gibson, Toxicol.Lett., 68(1-2):193(1993)). This nuclear receptor has been named theperoxisome proliferator-activated receptor (PPAR) and belongs to thesteroid nuclear receptors family that have a major effect on geneexpression and cell biology. Binding by peroxisome proliferators such asclofibrate, herbicides, and leukotriene antagonists with PPAR activatesthe nuclear receptor, which acts as a transcriptional factor, and cancause differentiation, cell growth and proliferation of peroxisomes.Although these agents are thought to play a role in hyperplasia andcarcinogenesis as well as altering the enzymatic capability of animalcells, such as rodent cells, these agents appear to have minimalnegative effects in human cells, as exemplfied by the safety of drugssuch as clofibrate (Green, Biochem. Pharm. 43(3):393(1992)).

Peroxisome proliferators typically contain a carboxylic functionalgroup. Therefore, PA, PB and various phenylacetic acid derivatives weretested for their ability to activate the PPAR and compared with knownperoxisomal proliferators. As shown in FIG. 22, Clofibrate, a knownactivator of peroxisosmal proliferation, caused a 4- to 5-fold increasein activation as measured by increased production of the responseelement for acyl-CoA oxidase, which is the rate limiting enzyme inbetaoxidation and is contained in peroxisomes (Dreyer et al., Biol.Cell, 77(1):67(1993)). PA and PB caused mild activation (double baselineactivity), naphthyl acetate was relatively more active (approximately2.5- to 4-fold increase) while the halogenated analogs of PB were verypotent stimulators. Interestingly, butyrate was not a significantperoxisomal proliferation activator.

The peroxisome proliferator-activated receptor has been shown to belongto the same family of nuclear receptors as the retinoid, thyroid andsteroid receptors and PPAR is known to interact with RXR, the receptorof 9-cis-retinoic acid (a metabolite of all-trans-retinoic acid).Because the PPAR signaling pathway converges with the 9-cis retinoidreceptor signal, it can be anticipated that retinoic acid or the likewill significantly enhance the activity of PA or PB or otherphenylacetic acid derivatives of this invention. Indeed, enhancement ofthe induction of HL-60 cell differentiation by NaPA in combination withretinoic acid is discussed above. Additionally, this synergisticresponse has been confirmed in other tumors, such as neuroblastoma,melanoma and rhabdomyosarcoma cells.

Thus, combination therapy consisting of administration (simultaneouslyin the same dosage form or simultaneously/sequentially in separatedosage forms) of Vitamin A, Vitamin D, Vitamin C, Vitamin E, B-carotene,of other retinoids and the like with PA, PB or other phenylacetic acidderivatives is encompassed by the instant invention for any of thetreatment regimes given herein. Appropriate doses of the phenylaceticacid derivatives include approximately 0.5-10 mM PA, more preferably0.5-5 mM PA, doses or equipotent doses of a pharmaceutically acceptablephenylacetic acid derivitive. Between 0.1 and 1.0 μM concentrations ofthe retinoids are expected to be effective. This combination therapyenhances, for instance, the efficacy of treatment with PA, PB or otherphenylacetic acid derivatives, taken alone, for cancer, anemia and AIDStreatment, wound healing, and treatment of nonmalignant disorders ofdifferentiation.

Agents affecting cellular peroxisomes have a major impact on oxidativestress and the redox state of a cell. Thus, further evidence that PA, PBor other phenylacetic acid derivatives activate PPAR can be found by therapid increase of gamma glutamyl transpeptidase and catalase followingcellular exposure to PA or PB as shown in FIG. 23. These antioxidantenzymes, whose activities are increased when peroxisome proliferationhas been activated, were increased by 100% 24 hours after administrationof sodium phenylbutyrate. This effect was reversed by approximately 48hours and activity was maintained below control levels through 100hours. The intracellular level of glutathione followed a similarbiphasic pattern with an initial increase (20%) followed by a fall tolevels below baseline at 100 h. The rapid induction with subsequentsharp decline of these antioxidant enzymes was observed in numeroustumor types from prostatic, breast and colon adenocarcinomas,osteosarcoma, and brain tumors. Nolecular analysis showed changes in therate of gene transcription of the GSH-related and antioxidant enzymes,which are consistent with activation of PPAR by PA, PB or their analogs.

Because peroxisomal enzymes are instrumental in defending againstoxidative stress, experiments were undertaken to examine the effects oftreatment with PA or PB on cells which were subjected to chemical orradiation stress. Pretreatment of glioblastomas (FIG. 42), breastcarcinoma and metastatic prostate cells with a non-toxic dose of PA orPB 72 hours prior to CO⁶⁰ γ-radiation or treatment with adriamycindemonstrated a significant dose-related increase in cell killing byeither modality. The surviving fraction of cells following drugtreatment was nearly one tenth the fraction surviving with nopretreatment, which suggests that PA, PB or other like analogs could beused to increase the efficacy of radiation therapy and chemotherapysubstantially. As such, the instant invention encompasses combinationanti-cancer therapy consisting of administration of an non-toxiceffective amount of phenylacetic acid or a pharmaceutically acceptablephenylacetic acid derivative (according to any of the dosageconcentration protocols given herein) in combination with radiationtherapy, particularly local treatment, or chemotherapy, particularlytargeted to the tumor cells. This adjuvant therapy can be administered,for instance, after approximately 24 hours, such as from 24 hours to 120hours or more, from the initiation of the administration of thederivative.

These results suggest a further consideration of a variety of pathogenicdisorders. Inflammatory response to tissue injury, pathogenesis ofemphysema, ischemia-associated organ injury (shock), doxorubicin-inducedcardiac injury, drug-induced hepatotoxicity, atherosclerosis, andhyperoxic lung injuries are each associated with the production ofreactive oxygen species and a change in the reductive capacity of thecell. Although long-term exposure to PA or other phenylacetic acidanalogs depletes cellular redox protection systems, short term treatmentwith PA and the like may have significant implications for treatment ofdisorders associated with increased reactive oxygen species.

SECTION J: USE OF PHENYLACETIC ACID AND ITS DERIVATIVES IN TREATMENT OFCANCERS HAVING A MULTIPLE-DRUG RESISTANT PHENOTYPE

In treating disseminated cancers, systemic treatment with cytotoxicagents is frequently considered the most effective treatment. However, anumber of cancers exhibit the ability to resist the cytotoxic effects ofthe specific antineoplastic drug administered as well as other agents towhich the patient's system has never been exposed. In addition, somecancers appear to have multiple drug resistance even prior to the firstexposure of the patient to an antineoplastic drug. Three mechanisms havebeen proposed to explain this phenomenon: P-glycoprotein Multiple DrugResistance (MDR), MDR due to Topoisomerase Poisons and MDR due toaltered expression of drug metabolizing enzymes (Holland et al., CancerMedicine, Lea and Febiger, Philadelphia, 1993, p. 618-622).

P-glycoprotein MDR resistance appears to be mediated by the expressionof an energy-dependent pump which rapidly removes cytotoxic agents fromthe cell. High levels of p-glycoprotein are associated withamplification of the MDR gene and transcriptional activation. Increasedexpression of p-glycoprotein can also be stimulated by heat shock, heavymetals, other cytotoxic drugs and liver insults, and ionizing radiationin some cell lines from some species. The results are not sufficientlyconsistent to confirm a causal relationship but are highly suggestive.

Topoisomerases are nuclear enzymes which are responsible for transientDNA strand breaks during DNA replication, transcription andrecombination. Cytotoxic agents, such as etoposide, doxorubicin,amsacrine and others are known poisons of topoisomerase II, and causelethal DNA strand breakage by the formation of stable complexes betweenthe DNA, topoisomerase II and drug. MDR to this type of drug is thoughtto be caused by changes in the nature and amount of enzymatic activity,which is thought to prevent the formation or effect of theDNA-enzyme-drug complex.

Some cytotoxic agents are able to induce increased metabolic capabilitywhich permits rapid elimination of the toxin. Among the enzymes whichhave been implicated are glutathione S-transferase isozymes (GSTs).These enzymes are responsible for the conjugation of the electrophilicmoieties of hydrophobic drugs with glutathione, which leads todetoxification and elimination of the drug.

As discussed above, PA and other phenylacetic acid analogs have beenshown to stimulate the proliferation of peroxisomes which contain someisozymes of GST. Based on that observation, it would be expected that PAand PB would also stimulate MDR. However, as shown in FIG. 25 it has nowbeen discovered that the opposite occurs. Thus, FIG. 25 shows theinhibition by PA of the growth of cells from a line of breast cancercells that exhibit the MDR phenotype. Up to 10 mM PA in cultures, growthof cells is dramatically inhibited in a dose-dependent manner.Surprisingly, PA and PB are more highly active againstadriamycin-resistant breast cancer cells than compared toadriamycin-sensitive cells. This increased sensitivity of the MDRphenotype is reproducible in other tumor models, including those thatare resistant to radiation therapy.

Thus, the instant invention provides a method of treating tumor cellpopulations in a patient that are resistant or able to survive currentconventional treatments, particularly tumors having a MDR phenotype, byadministration to the patient of non-toxic amounts of PA (such asamounts that provide up to 10 mM PA or an equipotent dose of apharmaceutically acceptable phenylacetic acid derivative) in thevicinity of the tumor or equivalently effective amounts of phenylaceticacid or a phenylacetic acid analog. PA or other analog dosage protocolssimilar to those described in relation to the potentiation ofdifferentiation in tumor cells by these phenylacetic acid-relatedcompounds, inlcuding the various combination therapies described herein,can be used to treat patients with resistant tumors such as MDR tumors.Long-term (weeks, months) or short-term (day(s)) substantiallycontinuous treatment regimens (including continuous administration orfrequent administration of separate doses) as well as pulsed regimens(days, weeks or months of substantially continuous administrationfollowed by a drug-free period) can beneficially be employed to treatpatients with MDR tumors.

SECTION K: PHENYLACETATE AND ITS DERIVATIVES, CORRELATION BETWEENPOTENCY AND LIPOPHILICITY

One potential problem that could hinder the clinical use ofphenylacetate is related to the large amounts of drug required toachieve therapeutic concentrations, i.e., over 300 mg/kg/day. Studieswere thus undertaken to develop analogs that are effective at lowerconcentrations. Studies in plants revealed that increasing thelipophilicity of a phenylacetate analogue (as measured by itsoctanol-water partition coefficient) enhanced its growth-regulatoryactivity [Muir, R. M., Fujita, T., and Hansch, C. Structure-activityrelationship in the auxin activity of mono-substituted phenylaceticacids. Plant Physiol., 42: 1519-1526, 1967.]. Calculated partitioncoefficient (CLOGP) was used to correlate the predicted lipophilicitywith the measured antitumor activity of phenylacetate analogues. Forthese analogues, enhanced potency in inducing cytostasis and phenotypicreversion in cultured prostate carcinoma, glioblastoma, and melanomacells was correlated with increased drug lipophilicity.

Cell Cultures.

Studies included the following humans tumor cell lines: (a)hormone-refractory prostatic carcinoma PC3, DU145, purchased from theAmerican Type Culture Collection (ATCC, Rockville, Md.); (b)glioblastoma U87, A172 (ATCC); (c) melanoma A375 and mel 1011, providedby J. Fidler (M. D. Anderson, Houston Tex.) and J. Weber (NCI, BethesdaMd.), respectively. Cells were maintained in RPMI 1640 supplemented with10% heat inactivated fetal calf serum (Gibco Laboratories), antibiotics,and 2 mM L-glutamine. Diploid human foreskin FS4 fibroblasts (ATCC), andhuman umbilical vein endothelial cells (HUVC) were used for comparison.The HUVC cells, isolated from freshly obtained cords, were provided byD. Grant and H. Kleinman (NIH, Bethesda Md.).

Antitumor Agents.

Sodium phenylacetate and phenylbutyrate were from Elan Pharmaceuticalcorp, Gainvesville Ga. Iodophenylacetate, 4-iodophenylbutyrate and4-chlorophenylbutyrate were synthesized by the Sandmeyer procedure fromthe corresponding 4-amino-phenyl-fatty acids. The halogenated productswere extracted from the acidic reaction mixtures with diethyl etherwhich was then taken to dryness. The residue was dissolved in boilinghexane and the crystals that formed on cooling were collected by suctionfiltration. The product was recrystallized from hexane until thereported melting points were obtained. Amides of phenylacetate andphenylbutyrate were produced by heating the sodium salts with a smallexcess of thionylchloride followed by the addition of ice-coldconcentrated ammonia. The amides were purified by recrystalization fromboiling water. The identity of synthesized compounds was verified bymelting point determination and by mass spectroscopy. All commerciallyavailable derivatives were purchased from Aldrich (Milwaukee, Wis.) orSigma (St. Louis, Mo.), depending on availability. Tested compounds wereall dissolved in distilled water, brought to pH 7.0 by the addition ofNaOH as needed, and stored in aliquots at -20° C. till used.

Calculation of Relative Drug Lipophilicities.

Estimation of the contribution of lipophilicity to the biologicalactivity of a molecule was based on its calculated logarithm ofoctanol-water partition coefficient (CLOGP). This was determined foreach compound using the BLOGP program of Bodor et al., (BLOGP version1.0, Center for Drug Discovery, University of Florida) assuming that thedegree of ionization is similar for all tested compounds.

Quantitation of Cell Growth and Viability.

Growth rates were determined by cell enumeration with a hemocytometerfollowing detachment with trypsin-EDTA, and by an enzymatic assay using3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertrazolium bromide (MTT).These two assays produced essentially the same results. Cell viabilitywas assessed by trypan blue exclusion.

Colony Formation in Semi-Solid Agar.

For analysis of anchorage independent growth, cells were harvested withtrypsin-EDTA and resuspended at 1.0×10⁴ cells per ml in growth mediumcontaining 0.36% agar (Difco). Two ml of cell suspension were added to60 mm plates (Costrar) which were pre-coated with 4 ml of solid agar(0.9%). Tested drugs were added at different concentrations, andcolonies composed of 30 or more cells were counted after 3 weeks.

Growth on Matrigel.

Cells were first treated with drugs in T.C. plastic dishes for 4-6 days,and then replated (5×10⁴ cells per well) onto 16 mm dishes (Costar,Cambridge, Mass.) coated with 250 ul of 10 mg/ml matrigel, areconstituted basement membrane (Collaborative Research). Drugs wereeither added to the dishes or omitted in order to determine thereversibility of effect. Net-like formation characteristic of invasivecells occurred within 12 hours, while invasion into the matrigel wasevident after 6-9 days.

Drug Uptake Studies.

Cells were plated in 6-well T.C. dishes (Costar) at 5×10⁵ cells perdish. The growth medium was replaced after 24 hrs with 750 ul of freshmedium containing 4.5×10⁵ DPM of either ¹⁴ C-phenylacetic acid (3.4mCi/mmol, Sigma) or ¹⁴ C-naphthylacetic acid (5.4 mCi/mmol, Sigma), andthe cultures were incubated for 10-180 minutes at 37° C. Labeling wasterminated by placing plates on ice. Cells were then washed twice with 5ml ice-cold phosphate buffer saline (PBS), detached by scraping, and theradioactivity retained by cells determined using liquid scintillation.Blank values were determined by incubating the radiolabled compounds inan empty dish.

Correlation Between Drug Lipophilicity and Growth-Inhibitory Effect ofPhenylacetate and its Analogues.

The growth inhibitory effect of these compounds on prostatic carcinoma,glioblastoma, and melanoma cell lines are expressed as IC₅₀ andcorrelated with drug lipophilicity determined using the CLOGP program.As seen in Tables 21 and 22, there is a good correlation betweencytostasis and lipophilicity. In agreement with previous observationswith phenylacetate (3), the cytostatic effect was selective as higherdrug concentrations were needed to significantly affect theproliferation of normal endothelial cells and skin fibroblasts. Nocytotoxicity (i.e., decline in cell viability) occurred during 4-6 daysof continuous treatment with the tested compounds.

                                      TABLE 21                                    __________________________________________________________________________    Phenylacetate and analogues containing alkyl-                                 chain substitutions: Relationship of IC.sub.50 to CLOGP                                    IC.sub.50 (mM)                                                                                   normal                                        Rx      CLOGP                                                                              prostate ca.                                                                        glioblastoma                                                                         melanoma                                                                            cells                                         __________________________________________________________________________    α-methoxy-PA                                                                    2.17 6     5.8    6     ND                                            PA      2.05 5     4.3    5     12                                            α-methyl-PA                                                                     2.42 2.6   3.8    3.5   12                                            α-ethyl-PA                                                                      2.77 2.1   2.8    2.2    9                                            PB      2.89 1     1.8    1     ND                                            4-chloro-PB                                                                           3.30 0.75  ND     0.8   ND                                            4-iodo-PB                                                                             3.85 0.36   0.27  0.22  ND                                            __________________________________________________________________________     ND, not determined                                                       

                                      TABLE 22                                    __________________________________________________________________________    Phenylacetate and analogues containing ring                                   substitutions: Relationship of IC.sub.50 to CLOGP                                          IC.sub.50 (mM)                                                                                   normal                                        Rx      CLOGP                                                                              prostate ca.                                                                        glioblastoma                                                                         melanoma                                                                            cells                                         __________________________________________________________________________    4-Hydroxy-PA                                                                          1.78 7.5   10     10    ND                                            PA      2.05 5     4.3    5     12                                            4-fluoro-PA                                                                           2.17 2.8   4      2.5   ND                                            2-methyl-PA                                                                           2.43 2.5   ND     ND    ND                                            3-methyl-PA                                                                           2.45X                                                                              2.1   ND     ND    ND                                            4-methyl-PA                                                                           2.47 2.1   ND     ND    ND                                            4-chloro-PA                                                                           2.48 1     0.9    1.2   3                                             3-chloro-PA                                                                           2.54 1.75  1.7    1.5   7                                             2-chloro-PA                                                                           2.56 2.4   2.1    2.5   ND                                            2,6-dichloro-PA                                                                       2.87 1     0.8    1     ND                                            4-iodo-PA                                                                             3.12 0.6   0.9    1.2   ND                                            1-naphtylacetate                                                                      3.16 0.8   0.9    0.8   2.8                                           __________________________________________________________________________     ND, not determined                                                       

Further analysis of structure-activity relationships was based on themethod of Hansch and Anderson used for the correlation of the anestheticand metabolic effects of barbiturates with their octanol-water partitioncoefficients [Hansch, C., and Anderson, S. M. The structure-activityrelationship in barbiturates and its similarity to that in othernarcotics. J. Med. Chem 10: 745-753, 1967.]. Adaptation of this methodassumes that, if the relationship is simple, it will follow theequation: log 1/C=slope log P+K. Plotting the log 1/IC₅₀ values obtainedwith prostatic cells vs drug CLOGP (FIG. 26) shows that the best fitline is described by the equation: log 1/IC₅₀ =0.89 CLOGP+0.55. Theslope of this line (0.89) is in the range of values found for theanesthetic potencies of a series of barbiturate analogues. Hansch andcolleagues also studied the effect of phenylacetate and its derivativeson plant growth. As shown in FIG. 27, the concentration range and rankorder of inhibition of plant growth by phenylacetate analogues arecomparable to the inhibition of growth of prostatic cancer cells by thissame series of compounds.

While the overall trend of enhanced activity of phenylacetatederivatives with increased lipophilicity is clear, some small deviationsoccur. For both chloro- and methyl-substitutions, the para position ismore potent than the ortho position. In addition, and despite theirnearly equal contributions to lipohilicity, para chloro-substitution wasmore potent than methyl. In contrast to derivatives containing ring oralpha-carbon substitutions, those with blocked carboxyl groups exhibiteda decline in cytostatic activity. The methyl ester of phenylacetate wasabout half as active than the free acid (IC₅₀ in DU145 prostatic cells8.8 mM versus 4.1 mM for phenylacetate). The amide forms were also lessactive than the parent compounds in this experimental system, with IC₅₀s of 2.0 mM for phenylbutyramide versus 1.2 mM for phenylbutyrate, and4.8 mM for phenylacetamide versus 4.1 mM for phenylacetate.

Drug Uptake.

One possible function of increasing lipophilicity is an increasing easewith which aromatic fatty acids can enter into, and cross the plasmamembrane as well as the membranes of other organelles. The rate ofphenylacetate uptake by tumor cells was compared that of the morehydrophobic analog, naphthylacetate (Table 22). After 10 minutes,relative to phenylacetate more than twice as much naphthylacetate hadentered the glioblastoma U87 cells (uptake of phenylacetic acid was 41%that of naphthylacetic acid) indicating that its movement through theplasma membrane was more than twice as fast as phenylacetate. After 20minutes, the amount of naphthylacetate taken up by the cells was as only26% greater than that of phenylacetate and at 180 minutes theintracellular levels of both compounds were nearly equal, suggestingthat at this time the more rapid influx of naphthylacetic acid wasbalanced by an equally rapid efflux. There was little further uptake andthe concentration of phenylacetate inside and outside the cells wasabout equal indicating that these cells do not actively accumulate mucharomatic fatty acid.

Phenotypic Reversion.

In addition to causing selective cytostasis, phenylacetate inducesmalignant cells to undergo reversion to a more benign phenotype. Theeffect of analogs on tumor biology was tested using as a model thehormone- refractory prostatic PC3 cells originally derived from a bonemetastasis. PC3 exhibit several growth characteristics in vitro thatcorrelate with their malignant behavior in vivo, includinganchorage-independent growth (i.e., colony formation in semi-solidagar), and formation of "net"-like structures when plated on areconstituted basement membrane (matrigel). The ability of phenylacetateand representative analogs to bring about loss of such properties issummarized in FIG. 27. Similar to the cytostatic effect, drug ability toinduce reversion to a non-malignant phenotype was highly correlated withthe calculated lipophilicity of the drugs. Of the tested compounds,naphthytacetate, as well as derivatives of phenylbutyrate andphenylacetate with iodo- and chlorine substitutions were found to be themost active on a molar basis. The relative efficacy of the compounds insuppressing anchorage independent growth was confirmed using U87glioblastoma cells (data not shown).

Discussion.

The comparative activity of phenylacetate and its analogues against anumber of tumor cell lines suggest that these compounds may form a newclass of therapeutic agents whose effectiveness varies with structure.Improved anticancer activity is achieved if factors controlling theiraction are understood, and toward this end the effects of systematicchanges in structure with changes in activity have been compared. Theoutstanding result is the discovery that: (a) there is a simplerelationship between the lipophilicity of a phenylacetate derivative andits activity against human tumor cells, and (b) the relative potencyobserved with human neoplasms is similar to that documented in plants,indicating that the role of the aromatic fatty acids in growthregulation has been conserved in evolution.

The efficacy of aromatic fatty acids was demonstrated in vitro usingtumor cell lines derived from patients with hormone-refractory prostaticcarcinoma, glioblastomas, and malignant melanoma. Like phenylacetate,several derivatives containing alpha-carbon or ring substitutions allinduced cytostasis and phenotypic reversion at non-toxic concentrations.Changes in tumor biology included reduction in cell proliferation rateand loss of malignant properties such as invasiveness andanchorage-independence. There were, however, significant differences inpotency. When compared to phenylacetate, analogs with naphthyl-,halogen- or alkyl-ring, as well as α-carbon alkyl substitutionsexhibited increased activity, while those with α-methoxy or hydroxylreplacement at the phenyl ring were less effective. Drug potency wascorrelated with the degree of calculated lipophilicity, indicating thatdifferences in efficacy may be due in part to the ease with which theseagents enter into and cross the lipid bilayer of cell membranes. Inagreement, uptake of the more hydrophobic compound, naphthylacetate, wassignificantly faster than that of phenylacetate. At equilibrium (about180 minutes for phenylacetate), however, there were no differences ineither the total intracellular concentration of both compounds, or thelevels inside and outside cells. These results suggest that the rates ofdrug uptake are balanced by proportional rates of efflux, and that theoverall capacity of the cell to retain such compounds is not muchgreater than that of the extracellular milieu.

Although there is a good correlation between drug potency andlipophilicity (see FIG. 26), small deviations within thephenylacetate-related series may give some clues regarding mechanisms ofaction. Halogen substitutions para to the alkylcarboxyl group were foundto increase potency more than those in the ortho position, suggestingthat orientation of the hydrophobic substituent may be important. At thepara position, chlorine had a greater impact on efficacy than a methylgroup despite nearly equal contributions to CLOGP, indicating thatelectronegativity may affect growth inhibitory interactions. Whileα-ethylphenylacetic acid, in which the carboxyl group is crowded by theadjacent ethyl group, was more potent than the parent compound, the morelipophilic analog α-methoxyphenylacetic acid was less active. Theα-methoxyphenylacetic acid is a significantly stronger acid, and thisgreater acidity could be important. Other parameters such as addition ofan aromatic ring to phenylacetate, or an increase in the distancebetween the aromatic nucleus and the carboxyl group did not causeanomalous enhancement or interference in biological activity(naphthylacetate and phenylbutyrate were about as active as would beexpected on the basis of their lipophilicity). The importance of a freecarboxyl group is unclear. The amide forms of phenylacetate andphenylbutyrate, in which the carboxylic group is blocked, were lesscytostatic compared to the parental compounds and failed to induce celldifferentiation (unpublished data). Moreover, phenylacetylglutamine hasno detectable effect on cell growth and maturation. It appears,therefore, that a free carboxyl group may be essential for some aspectsof the antitumor activity of phenylacetate and derivatives.

The correlation between partition coefficients and bioactivity of thearomatic fatty acids is reminiscent of that observed for a large numberof other lipophilic agents. A survey by Hansch and Anderson revealedthat, in a variety of animal tissues, the anesthetic and metaboliceffects of barbiturates corresponded well with their hydrophilicity,having an average slope of about 1 compared to a slope of about 0.67 forlipophilic interaction with protein. It was concluded that the criticalstep in initiating biological activity was entry into the lipid bilayer,probably followed by interaction with membrane proteins. Some of thesubsequently identified targets of barbiturates are indeed, membraneproteins and these include the GABA receptor-chloride in neurons, theATP-K⁺ pump in pancreatic B-cells, and the G-protein that stimulates PLCactivity in leukemic cells. Despite a wide body of literatureimplicating phenylacetate and analogs in growth control throughoutphylogeny, little is known regarding their mode of action. In plants,phenylacetate and naphtylacetate are endogenous growth hormones (auxins)known to stimulate proliferation at micromolar concentrations, whileinhibiting growth at millimolar levels. As growth inhibitors (but notstimulators), the effect of phenylacetate analogues on rapidlydeveloping embryonic plant tissues, like that on human tumor cells, is asimple function of their lipophilicities. These similarities in potency,summarized in FIG. 27, suggest that some of the underlying mechanisms ofnegative growth control may be similar as well.

There is accumulating evidence indicating that phenylacetate andderivatives may act through multiple mechanisms to alter gene expressionand cell biology. At growth inhibitory concentrations, the aromaticfatty acids could alter the pattern of DNA methylation, an epigeneticmechanism controlling the transcription of various eukaryotic genes.Phenylacetate inhibits DNA methylation in plant and mammalian cells, andboth phenylacetate and phenylbutyrate were shown to activate theexpression of otherwise dormant methylation-dependent genes. DNAhypomethylation per se is not sufficient to induce gene expression.Preliminary findings indicate that phenylacetate, phenylbutyrate andseveral analogs activate a nuclear receptor that functions as atranscriptional factor; interestingly, the receptor is a member of asteroid nuclear receptor superfamily, the ligands of which arecarboxylic acids and include well characterized differentiation inducerssuch as retinoids.

In addition to affecting gene transcription, the phenyl-fatty acids mayinterfere with protein post-translational processing by inhibiting themevalonate (MVA) pathway of cholesterol synthesis. MVA is a precursor ofseveral isopentenyl moieties required for progression through the cellcycle, and of prenyl groups that modify a small set of criticalproteins. The latter include plasma membrane G and G-like proteins(e.g., ras) involved in mitogenic signal transduction (molecular weight20-26 kDa), and nuclear envelope lamins that play a key role in mitosis(44-74 kDa). The aromatic fatty acids can conjugate with coenzyme-A,enter the pathway to chain elongation, and interfere with lipidmetabolism in general. Furthermore, compounds such as phenylacetate canassume a conformation resembling mevalonate pyrophosphate and inhibitMVA utilization specifically. It was recently demonstrated thatphenylacetate activity against poorly differentiated mammalian tissues(human glioblastoma cells and the developing fetal brain) is associatedwith inhibition of MVA decarboxylation and a decline in proteinisoprenylation. Rapidly developing mammalian and plant tissues arehighly dependent upon MVA for cell replication. Inhibition of MVAutilization by phenylacetate-related compounds could thus be responsiblein part for their effect documented in such highly divergent organisms.

In conclusion, phenylacetate and analogs appear to represent a new classof pleiotropic growth regulators that might alter tumor cell biology byaffecting gene expression at both the transcriptional and posttranscriptional levels. Phenylacetate and phenylbutyrate have alreadybeen established as safe and effective in treatment of hyperammonemia,and phase I clinical trials in adults with cancer confirmed thatmillimolar levels can be achieved in the plasma and cerebrospinal fluidwith no significant toxicities (discussed herein). However, rather largedoses (300 mg/kg/day or more) are required to achieve potentiallytherapeutic levels. The identified relationship between lipophilicity ofcommercially available analogs and their antitumor activity inexperimental models led us to predict that analogs with greater CLOGPs,e.g., iodo derivatives of phenylacetate and phenylbutyrate, would behighly effective. Indeed, these compounds were found to be the mostpotent aromatic fatty acids yet tested. With this approach, it should bepossible to identify highly effective and safe antitumor agents suitablefor clinical application.

Modes of Drug Administration

NaPA (or PAA derivatives) may be administered locally or systemically.Systemic administration means any mode or route of administration whichresults in effective levels of active ingredient appearing in the bloodor at a site remote from the site of administration of said activeingredient.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for intravenous, intramuscular,subcutaneous, oral, nasal, enteral, parenteral or topicaladministration. In some cases, a combination of types of formulationsmay be used simultaneously to achieve systemic administration of theactive ingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, dragees, pills, tablets (including coated tablets),elixirs, suspensions, and syrups or inhalations.

Solid dosage forms in addition to those formulated for oraladministration include rectal suppositories.

The compounds of the present invention may also be administered in theform of an implant.

Suitable formulations for topical administration include creams, gels,jellies, mucilages, pastes and ointments.

Suitable injectable solutions include intravenous, subcutaneous, andintramuscular injectable solutions. The compounds of the presentinvention may also be administered in the form of an infusion solutionor as a nasal inhalation or spray.

The compounds of the present invention may also be used concomitantly orin combination with selected biological response modifiers, e.g.,interferons, interleukins, tumor necrosis factor, glutamine antagonists,hormones, vitamins, as well as anti-tumor agents and hematopoieticgrowth factors, discussed above.

It has been observed that NaPA is somewhat malodorous. Therefore, it maybe preferable to administer this compound in the presence of any of thepharmaceutically acceptable odor-masking excipients or as its precursorphenylbutyrate (or a derivative or analog thereof) which has nooffensive odor.

The PAA and its pharmaceutically acceptable derivatives to be used asantitumor agents can be prepared easily using pharmaceutical materialswhich themselves are available in the art and can be prepared byestablished procedures. The following preparations are illustrative ofthe preparation of the dosage forms of the present invention, and arenot to be construed as a limitation thereof.

EXAMPLE 21

Parenteral Solution 1

A sterile aqueous solution for parenteral administration containing 200mg/ml of NaPA for treating a neoplastic disease is prepared bydissolving 200 g. of sterilized, micronized NaPA in sterilized NormalSaline Solution, qs to 1000 ml. The resulting sterile solution is placedinto sterile vials and sealed. The above solution can be used to treatmalignant conditions at a dosage range of from about 100 mg/kg/day toabout 1000 mg/kg/day. Infusion can be continuous over a 24 hour period.

EXAMPLE 22

Parenteral Solution 2

A sterile aqueous solution for parenteral administration containing 50mg/ml of NaPA is prepared as follows:

    ______________________________________                                        Ingredients           Amount                                                  ______________________________________                                        NaPA, micronized      50 g.                                                   Benzyl alcohol        0.90% w/v                                               Sodium chloride       0.260% w/v                                              Water for injection, qs                                                                             1000 ml                                                 ______________________________________                                    

The above ingredients, except NaPA, are dissolved in water andsterilized. Sterilized NaPA is then added to the sterile solution andthe resulting solution is placed into sterile vials and sealed. Theabove solution can be used to treat a malignant condition byadministering the above solution intravenously at a flow rate to fallwithin the dosage range set forth in Example 21.

EXAMPLE 23

Parenteral Solution 3

A sterile aqueous solution for parenteral administration containing 500mg/ml of sodium phenylbutyrate is prepared as follows:

    ______________________________________                                        Ingredients           Amount                                                  ______________________________________                                        Sodium phenylbutyrate 500 g.                                                  Dextrose              0.45% w/v                                               Phenylmercuric nitrate                                                                              0.002% w/v                                              Water for injection, qs                                                                             1000 ml.                                                ______________________________________                                    

The preparation of the above solution is similar to that described inExamples 21 and 22.

EXAMPLE 24

Tablet Formulation 1

A tablet for oral administration containing 300 mg of NaPA is preparedas follows:

    ______________________________________                                        Ingredients            Amount                                                 ______________________________________                                        NaPA                   3000 g.                                                Polyvinylpyrrolidone   225 g.                                                 Lactose                617.5 g                                                Stearic acid           90 g.                                                  Talc                   135 g.                                                 Corn starch            432.5 g.                                               Alcohol                45 L                                                   ______________________________________                                    

NaPA, polyvinylpyrrolidone and lactose are blended together and passedthrough a 40-mesh screen. The alcohol is added slowly and thegranulation is kneaded well. The wet mass is screened through a 4-meshscreen, dried overnight at 50° C. and screened through a 20-mesh screen.The stearic acid, talc and corn starch is bolted through 60-mesh screenprior to mixing by tubing with the granulation. The resultinggranulation is compressed into tablets using a standard 7/16 inchconcave punch.

EXAMPLE 25

Tablet Formulation 2

A tablet for oral administration containing 200 mg of sodiumphenylbutyrate is prepared as follows:

    ______________________________________                                        Ingredients             Amount                                                ______________________________________                                        Sodium phenylbutyrate   2240 g.                                               Compressible sugar (Di-Pac)                                                                           934 g.                                                Sterotex                78 g.                                                 Silica gel (Syloid)     28 g.                                                 ______________________________________                                    

The above ingredients are blended in a twin-shell blender for 15 minutesand compressed on a 13/22 inch concave punch.

EXAMPLE 26

Intranasal Suspension

A 500 ml sterile aqueous suspension is prepared for intranasalinstallation as follows:

    ______________________________________                                        Ingredients             Amount                                                ______________________________________                                        NaPA, micronized        30.0 g.                                               Polysorbate 80          2.5 g.                                                Methylparaben           1.25 g.                                               Propylparaben           0.09 g.                                               Deionized water, qs 500 ml                                                    ______________________________________                                    

The above ingredients, with the exception of NaPA, are dissolved inwater and sterilized by filtration. Sterilized NaPA is added to thesterile solution and the final suspensions are aseptically filled intosterile containers.

EXAMPLE 27

Ointment

An ointment is prepared from the following ingredients:

    ______________________________________                                        Ingredients      Amount                                                       ______________________________________                                        NaPA             10 g.                                                        Stearyl alcohol   4 g.                                                        White wax         8 g.                                                        White petrolatum 78 g.                                                        ______________________________________                                    

The stearyl alcohol, white wax and white petrolatum are melted over asteam bath and allowed to cool. The NaPA is added slowly to the ointmentbase with stirring.

EXAMPLE 28

Lotion

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        Sodium phenylbutyrate    1.00 g.                                              Stearyl methylcellulose (4,500)                                               Solution (2%)            25.00 ml                                             Benzalkonium chloride    0.03 g.                                              Sterile water           250.00 ml                                             ______________________________________                                    

The benzalkonium chloride is dissolved in about 10 ml. of sterile water.The sodium phenylbutyrate is dispersed into methylcellulose solution bymeans of vigorous stirring. The methylcellulose (4,500) used is a highviscosity grade. The solution of benzalkonium chloride is then addedslowly while stirring is continued. The lotion is then brought up to thedesired volume with the remaining water. Preparation of the lotion iscarried out under aseptic conditions.

EXAMPLE 29

Dusting Powder

    ______________________________________                                        Ingredients         Amount                                                    ______________________________________                                        NaPA                25 g.                                                     Sterilized absorbable maize                                                                       25 g.                                                     starch BP dusting powder                                                      ______________________________________                                    

The dusting powder is formulated by gradually adding the sterilizedabsorbable dusting powder to NaPA to form a uniform blend. The powder isthen sterilized in conventional manner.

EXAMPLE 30

Suppository, Rectal and Vaginal Pharmaceutical Preparations

Suppositories, each weighing 2.5 g. and containing 100 mg. of NaPA areprepared as follows:

    ______________________________________                                        Ingredients      Amount/1000                                                  ______________________________________                                        Suppositories    100 g.                                                       NaPA, micronized                                                              Propylene glycol 150 g.                                                       Polyethylene glycol                                                                            2500 g.                                                      4000, qs                                                                      ______________________________________                                    

NaPA is finely divided by means of an air micronizer and added to thepropylene glycol and the mixture is passed through a colloid mill untiluniformly dispersed. The polyethylene glycol is melted and the propyleneglycol dispersion added slowly with stirring. The suspension is pouredinto unchilled molds at 40° C. Composition is allowed to cool andsolidify and then removed from the mold and each suppository is foilwrapped.

The foregoing suppositories are inserted rectally or vaginally fortreating neoplastic disease.

It is known that intracellular glutathione plays a major role indetoxification and repair of cellular injury by chemical and physicalcarcinogens. NaPA treatment of normal or tumor cells markedly inducedthe activity of intracellular glutathione approximately 2-10 folddepending on growth conditions. Nontoxic agents that can induceglutathione are highly desirable since these are likely to protect cellsfrom damage by a variety of chemical carcinogens and ionizing radiation.

Taken together, the present invention demonstrates that NaPA, NaPB andother PAA derivatives have valuable potential in cancer prevention incase such as high risk individuals, for example, heavy smokers withfamilial history of lung cancer, inherited disorders of concogeneabnormalities (Li-Fraumeni syndrome), individuals exposed to radiation,and patients in remission with residual disease. Furthermore, thesecompounds can be used in combination with other therapeutic agents, suchas chemicals and radiation, to enhance tumor responses and minimizeadverse effects such as cytotoxicity and carcinogenesis. The antitumoractivity, lack of toxicity, and easy administration qualify NaPA as apreferred chemopreventive drug.

I claim:
 1. A method of inducing the differentiation of a cellcomprising administering to the cell a differentiation inducing amountof a compound of the formula I: ##STR3## wherein R₀ is aryl, phenoxy,substituted aryl or substituted phenoxy;R₁ and R₂ are, independently, H,hydroxy, lower alkoxy, lower straight or branched chain alkyl orhalogen; R₃ and R₄ are, independently, H, lower alkoxy, lower straightor branched chain alkyl or halogen; and n is an integer from 0 to 2;pharmaceutically-acceptable salt thereof or a mixture thereof.
 2. Themethod of claim 1, wherein the compound is sodium phenylacetate.
 3. Themethod of claim 1, wherein the compound is sodium phenylbutyrate.
 4. Themethod of claim 1, wherein the differentiation inducing amount is from50 to 1000 mg/kg/day.
 5. The method of claim 1, wherein thedifferentiation inducing amount is from 300 to 500 mg/kg/day.
 6. Themethod of claim 1, wherein the differentiation inducing amount is from150 to 250 mg/kg/day.
 7. The method of claim 1, wherein R₀ is aryl orphenoxy, the aryl and phenoxy being unsubstituted or substituted with,independently, one or more halogen, hydroxy or lower alkyl;R₁ and R₂ areindependently H, lower alkoxy, hydroxy, lower alkyl or halogen; and R₃and R₄ are independently H, lower alkyl, lower alkoxy or halogen;apharmaceutically-acceptable salt thereof, or a mixture thereof.
 8. Themethod of claim 1, whereinR₀ is phenyl, naphthyl, or phenoxy, thephenyl, naphthyl and phenoxy being unsubstituted or substituted with,independently, one or more moieties of halogen, hydroxy or lower alkyl.9. The method of claim 1, whereinR₀ is phenyl, naphthyl, or phenoxy, thephenyl, naphthyl and phenoxy being unsubstituted or substituted with,independently, from 1 to 4 moieties of halogen, hydroxy or lower alkylof from 1 to 4 carbon atoms; R₁ and R₂ are, independently, H, hydroxy,lower alkoxy of from 1 to 2 carbon atoms, lower straight or branchedchain alkyl of from 1 to 4 carbon atoms or halogen; and R₃ and R₄ are,independently, H, lower alkoxy of from 1 to 2 carbon atoms, lowerstraight or branched chain alkyl of from 1 to 4 carbon atoms or halogen.10. The method of claim 1, wherein n is 0; R₀ is aryl or substitutedaryl; R₁ and R₂ are H, lower alkoxy, or lower alkyl;pharmaceutically-acceptable salts thereof, or mixtures thereof.
 11. Themethod of claim 1, wherein the compound is α-methylphenylacetic acid,α-ethylphenylacetic acid, α-hydroxyphenylacetic acid,α-methoxyphenylacetic acid, 1-naphthylacetic acid, 4-chlorophenylaceticacid, 4-iodophenylacetic acid, 4-fluorophenylacetic acid,3-chlorophenylacetic acid, 2-chlorophenylacetic acid,2,6-dichlorophenylacetic acid, 2-methylphenylacetic acid,3-methylphenylacetic acid, 4-methylphenylacetic acid, phenoxypropionicacid, 4-chlorophenylbutyric acid, 4-iodophenylbutyric acid,4-fluorophenylbutyric acid, 3-chlorophenylbutyric acid, or2-chlorophenylbutyric acid.
 12. The method of claim 1, wherein thecompound is administered topically.
 13. The method of claim 1, whereinthe compound is applied at a concentration of from about 0.1 mM to about10 mM.
 14. The method of claim 1, wherein the compound is administeredocularly.
 15. The method of claim 1, wherein the compound isadministered orally.
 16. The method of claim 1, wherein the compound isadministered in the form of a suppository.
 17. The method of claim 1,wherein the compound is administered parenterally.
 18. The method ofclaim 1, wherein the compound is administered intermittently.
 19. Themethod of claim 1, wherein the compound is administered continuously.20. The method of claim 1, wherein the cell is a prostatic carcinomacell, a glioma cell, an astrocytoma cell, an adenocarcinoma cell, amelanoma cell, a lymphoma cell, a leukemia cell, a breast cancer cell,an osteosarcoma cell, a fibrosarcoma cell, a squamous cancer cell, aneuroblastoma cell, a non-small cell lung cancer cell, a mesotheliomacell, a multiple myeloma cell, a renal carcinoma cell, a Kaposi'ssarcoma cell, or a Lennert's T-Cell lymphoma cell.
 21. The method ofclaim 1, wherein the cell is a benign hyperplastic prostatic cell, apapilloma cell, or a myelodisplasic cell.